Chicken Primordial Germ Cells Research Articles

Comparison of the MicroRNA Expression Profiles of Male and Female Avian Primordial Germ Cell Lines.

Primordial germ cells (PGCs) are the precursors of adult germ cells, and among the embryonic stem-like cells in the bird embryo, only they can transmit the genetic information to the next generation. Despite the wide range of applications, very little is known about the mechanism that governs primordial germ cell self-renewal and differentiation. As a first step, we compared 12 newly established chicken PGC lines derived from two different chicken breeds, performing CCK-8 proliferation assay. All of the lines were derived from individual embryos. A significant difference was found among the lines. As microRNAs have been proved to play a key role in the maintenance of pluripotency and the cell cycle regulation of stem cells, we continued with a complex miRNA analysis. We could discover miRNAs expressing differently in PGC lines with high proliferation rate, compared to PGC lines with low proliferation rate. We found that gga-miR-2127 expresses differently in female and male cell lines. The microarray analysis also revealed high expression level of the gga-miR-302b-3p strand (member of the miR-302/367 cluster) in slowly proliferating PGC lines compared to the gga-miR-302b-5p strand. We confirmed that the inhibition of miR-302b-5p significantly increases the doubling time of the examined PGC lines. In conclusion, we found that gga-miR-181-5p, gga-miR-2127, and members of the gga-miR-302/367 cluster have a dominant role in the regulation of avian primordial germ cell proliferation.

NICD-mediated notch transduction regulates the different fate of chicken primordial germ cells and spermatogonial stem cells

BackgroundNotch signaling is mainly regulated by Notch1 during development of chicken germ stem cells; however, the molecular mechanisms that contribute to generation of these germ stem cells have not been thoroughly investigated.ResultsIn our studies, Overexpression of the Notch1 NICD promoted development of the reproductive ridge, but inhibited the formation of seminiferous tubules. The formation efficiency of PGCs in the reproductive ridge following overexpression of NICD (7.5% ± 0.11) was significantly higher than that (4.9% ± 0.17, p < 0.05) following inhibition of NICD, While the formation efficiency of spermatogonial stem cells (SSCs) in the testes (12.7% ± 0.08) was significantly lower after NICD overexpression than that after inhibition of NICD (16.3% ± 0.16, p < 0.05). Using co-immunoprecipitation, we found that this anomaly stemmed from the reversal of dissociation of the Notch-regulated transcription factor CBF-1/RBP co-suppression complex during the differentiation of PGCs into SSCs. This dissociation of the CBF-1/RBP co-suppressing complex during the differentiation of ESCs into PGCs resulted in the release of HDAC1 and HDAC2 and the recruitment of mastermind-like 1 to form a coactive complex to promote the expression of the downstream transcription suppressor hairy/enhancer of split-1. Dynamic expression of transducin-like enhancer of split 3, TLE4, and C-terminal binding protein 2 during further differentiation of PGCs inhibited the dissociation of the CBF-1/RBP co-suppression complex and inhibited the expression of the downstream genes.ConclusionsIn summary, Notch signaling plays diametrically opposing roles during normal development of chicken PGCs and SSCs, and these functions was determined by the expression of NICD, changes in the CBF-1/RBP complex composition, and histone modification.

Long-term in vitro culture and preliminary establishment of chicken primordial germ cell lines.

Primordial germ cells (PGCs) are precursors of functional gametes and can be used as efficient transgenic tools and carriers in bioreactors. Few methods for long-term culture of PGCs are available. In this study, we tested various culture conditions for PGCs, and used the optimum culture system to culture chicken gonad PGCs for about three hundred days. Long-term-cultured PGCs were detected and characterized by karyotype analysis, immunocytochemical staining of SSEA-1, c-kit, Sox2, cDAZL, and quantitative RT-PCR for specific genes like Tert, DAZL, POUV, and NANOG. Cultured PGCs labeled with PKH26 were reinjected into Stage X recipient embryos and into the dorsal aorta of Stage 14–17 embryos to assay their ability of migration into the germinal crescent and gonads, respectively. In conclusion, the most suitable culture system for PGCs is as follows: feeder layer cells treated with 20 μg/mL mitomycin C for 2 hours, and with 50% conditioned medium added to the factor culture medium. PGCs cultured in this system retain their pluripotency and the unique ability of migration without transformation, indicating the successful preliminary establishment of chicken primordial germ cell lines and these PGCs can be considered for use as carriers in transgenic bioreactors.

Generation of haploid spermatids from chicken embryonal primordial germ cells.

In vitro production of functional spermatids has special significance in the research of spermatogenesis and the treatment of male infertility. Primordial germ cells (PGCs) are the precursors of oocyte and sperm, which generate the totipotent cells. Studies have shown that PGCs have the potential ability to develop meiotic spermatids in vitro. Here we have shown that retinoic acid (RA) leads to PGC differentiation, and SCF can improve the efficiency of induction. We indicate an efficient approach to produce haploid spermatids from chicken PGCs in the presence of RA and stem cell factor (SCF). Real-time RT-PCR assays showed that RA and SCF induced a remarkable increase in expression of SYCP1, ACR, BOULE and DCM1 of meiotic germ cells and haploid germ cells, respectively. DNA content assays revealed that RA and SCF induced a remarkable increase of haploid cells. This study provides a theoretical basis and a great animal model for spermatogenesis study.

Effectiveness of DMSO concentration on recovery rate and viability of primordial germ cell of Gaok chicken

&lt;p class="abstrak2"&gt;Recent technological developments to produce germ line chimeras with primordial germ cell (PGC) transfer into the recipient embryo provide an opportunity to conserve and retrieval of chicken genetic resources in complete form. The study was conducted to obtain the most effective DMSO percentage to recovery rate and viability of Gaok chicken PGC after freezing which will later be feasible to be transferred. In this study, the eggs of Gaok chicken were incubated for about 2.5 - 3 days to obtain embryos at stages 14 - 16. Blood retrieval was done through the dorsal aorta using micropipettes under microscope. The procedure of PGC isolation of Gaok chicken with centrifugation gradient was using nycodenz as a substance. Commercially available cryoprotectants (dimethyl sulfoxide = DMSO) were used for PGC freezing. Isolated and frozen PGCs of Gaok chicken were diluted with cryoprotectants containing 2.5; 5 and 10% DMSO in fetal bovine serum (FBS). The recovery rate of 2.5; 5 and 10% DMSO concentration were 36.4; 48.2 and 48 % respectively. The viability of PGC after freezing was significantly higher for 5% DMSO compared with 2.5% DMSO (P&amp;lt;0.05), but not different from 10% DMSO. It can be concluded that the concentration DMSO of 5 % was effectve contration in freezing Gaok chicken PGC.&lt;/p&gt;

Chicken NANOG self-associates via a novel folding-upon-binding mechanism.

NANOG plays a pivotal role in pluripotency acquisition and lineage specification in higher vertebrates, and its expression is restricted to primordial germ cells (PGCs) during early embryonic development. Mammalian NANOG self-associates via conserved tryptophan-repeat motifs in the C-terminal domain (CTD) to maintain pluripotency. Avian NANOG, however, lacks the conserved motifs, and the molecular mechanism underlying the biologic function is not clearly understood. Here, using spectroscopic and biochemical methods as well as cell-based assays, we report that chicken NANOG (cNANOG) oligomerizes through its CTD via a novel folding-upon-binding mechanism. The CTD of cNANOG is disordered as a monomer and associates into an α-helical multimer driven by intermolecular hydrophobic interactions. Mutation of key aromatic residues in the CTD abrogates the self-association, leading to a loss of the proliferation of chicken PGCs and blastoderm cells. Our results demonstrate that the CTD of cNANOG belongs to a novel IDP that switches into a helical oligomer via self-association, enabling the maintenance of PGCs and blastoderm cells.-Choi, H. J., Kim, I., Lee, H. J., Park, Y. H., Suh, J.-Y., Han, J. Y. Chicken NANOG self-associates via a novel folding-upon-binding mechanism.

The early development of germ cells in chicken.

Primordial germ cells (PGCs) are the founder cells for mature gametes, the vehicles by which individuals transmit genetic and epigenetic information to later generations. Since the 19th century, avian species (chickens in particular) have been widely used for germ cell research. Previous studies have used chicken PGCs for a variety of research applications, including as a model for studies focusing on germline development. Other applications of chicken PGCs, including conservation efforts for avian species and methods of producing transgenic birds, have further reinforced the importance of these cells. However, much remains to be revealed about the origin and role of PGCs during their development in the chicken. Here, we provide a comprehensive review of chicken PGCs, focusing in particular upon their initial profiles and physiological changes during development as regulated by environmental factors and/or intrinsic mechanisms. We also emphasise sex-dependent differences in PGC development after settlement within the gonads, as well as future applications for avian PGCs.

Retinoic acid promotes expression of germline-specific genes in chicken blastoderm cells by stimulating Smad1/5 phosphorylation in a feeder-free culture system

BackgroundProducing transgenic chickens with chicken blastodermal cells (cBCs) is inefficient due to the extremely low germline transmission capacity of cBCs. As chicken primordial germ cells (PGCs) have been reported as an efficient method for producing transgenic chickens, the inefficiency of cBCs could potentially be resolved by inducing them to differentiate into germ cells. However, whether chemical inducers are able to enhance cBCs germline competence in vitro is unknown and the molecular mechanisms of differentiation of chicken pluripotent cells into germ cells are poorly understood.ResultsWe cultured cBCs with a monolayer morphology in E8 medium, a xeno- and feeder-free medium. We showed that retinoic acid (RA) treatment increased expression of germ cell-specific genes in cBCs. Using western blot, we determined that RA stimulated Smad1/5 phosphorylation. Moreover, Smad1/5 activation regulates the expression of germ cell-specific genes, as co-treatment with a Smad1/5 phosphorylation inhibitor or activator alters expression of these genes. We also demonstrate that Smad1/5 is required for RA-induced differentiation by RNA interference knockdown.ConclusionOur results demonstrated that E8 medium is able to maintain cBC growth for weeks and RA treatment induced germ cell differentiation of cBCs through the BMP-Smad1/5 signaling pathway.

Chicken Model for Inducing Primordial Germ Cells Differentiating into Motile Spermsin vitro

Stem cell technologies have been widely used in the study of spermatogenesis. However, deriving motile sperm from stem cells in vitro is still rarely achieved. We found that chicken primordial germ cells could directly differentiate into sperm by using retinoic acid in a non-testicular culture system. The induced sperms were characterised by RT-PCR, immunofluorescence and flow cytometry techniques. Results suggested that chicken primordial germ cells could produce motile sperm in vitro. Our work has provided a novel animal model of spermatogenesis in vitro, which might be used for male reproductive mechanism research.

Regulatory elements and transcriptional control of chicken vasa homologue (CVH) promoter in chicken primordial germ cells

BackgroundPrimordial germ cells (PGCs), the precursors of functional gametes, have distinct characteristics and exhibit several unique molecular mechanisms to maintain pluripotency and germness in comparison to somatic cells. They express germ cell-specific RNA binding proteins (RBPs) by modulating tissue-specific cis- and trans-regulatory elements. Studies on gene structures of chicken vasa homologue (CVH), a chicken RNA binding protein, involved in temporal and spatial regulation are thus important not only for understanding the molecular mechanisms that regulate germ cell fate, but also for practical applications of primordial germ cells. However, very limited studies are available on regulatory elements that control germ cell-specific expression in chicken. Therefore, we investigated the intricate regulatory mechanism(s) that governs transcriptional control of CVH.ResultsWe constructed green fluorescence protein (GFP) or luciferase reporter vectors containing the various 5′ flanking regions of CVH gene. From the 5′ deletion and fragmented assays in chicken PGCs, we have identified a CVH promoter that locates at −316 to +275 base pair fragment with the highest luciferase activity. Additionally, we confirmed for the first time that the 5′ untranslated region (UTR) containing intron 1 is required for promoter activity of the CVH gene in chicken PGCs. Furthermore, using a transcription factor binding prediction, transcriptome analysis and siRNA-mediated knockdown, we have identified that a set of transcription factors play a role in the PGC-specific CVH gene expression.ConclusionsThese results demonstrate that cis-elements and transcription factors localizing in the 5′ flanking region including the 5′ UTR and an intron are important for transcriptional regulation of the CVH gene in chicken PGCs. Finally, this information will contribute to research studies in areas of reproductive biology, constructing of germ cell-specific synthetic promoter for tracing primordial germ cells as well as understanding the transcriptional regulation for maintaining germness in PGCs.

Efficient TALEN-mediated gene targeting of chicken primordial germ cells.

In this work we use TALE nucleases (TALENs) to target a reporter construct to the DDX4 (vasa) locus in chicken primordial germ cells (PGCs). Vasa is a key germ cell determinant in many animal species and is posited to control avian germ cell formation. We show that TALENs mediate homology-directed repair of the DDX4 locus on the Z sex chromosome at high (8.1%) efficiencies. Large genetic deletions of 30 kb encompassing the entire DDX4 locus were also created using a single TALEN pair. The targeted PGCs were germline competent and were used to produce DDX4 null offspring. In DDX4 knockout chickens, PGCs are initially formed but are lost during meiosis in the developing ovary, leading to adult female sterility. TALEN-mediated gene targeting in avian PGCs is therefore an efficient process.

C-X-C chemokine receptor type 4 (CXCR4) is a key receptor for chicken primordial germ cell migration.

In mammals, germ cells originate outside of the developing gonads and follow a unique migration pattern through the embryonic tissue toward the genital ridges. Many studies have attempted to identify critical receptors and factors involved in germ cell migration. However, relatively few reports exist on germ cell receptors and chemokines that are involved in germ cell migration in avian species. In the present study, we investigated the specific migratory function of C-X-C chemokine receptor type 4 (CXCR4) in chicken primordial germ cells (PGCs). We induced loss-of-function via a frameshift mutation in the CXCR4 gene in chicken PGCs using clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR/Cas9) genome editing. The migratory capacity of CXCR4 knockout PGCs was significantly reduced in vivo after transplantation into recipient embryos. However, CXCR4-expressing somatic cell lines, such as chicken DT40 and DF1, failed to migrate into the developing gonads, suggesting that another key factor(s) is necessary for targeting and settlement of PGCs into the genital ridges. In conclusion, we show that CXCR4 plays a critical role in the migration of chicken germ cells.

Genome Editing Mediated by Primordial Germ Cell in Chicken.

Rapid development of genome editing technology has facilitated the studies on exploring specific gene functions and establishment of model animals. In livestock, the technology has contributed to create high value in industry fields, e.g., enhancing productivity or acquiring the resistance against disease. Meanwhile, genome editing in avian species has been emphasized because of their applicable possibilities in terms of highly productive chickens, disease-controlled avian lines, and development of novel biological models. Induction of exogenous gene using virus system or transposition in chicken primordial germ cells (PGCs) has been widely used for producing transgenic chicken, and recently developed programmable genome editing (PGE) technologies such as tale transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas9) are expected to maximize the applicable potentials of avian species. In this regard, this chapter will cover the methods for producing genome-edited chicken by piggyBac transposition and gene targeting technology, TALEN, and CRISPR/Cas9.

Avian Primordial Germ Cells.

Germ cells transmit genetic information to the next generation through gametogenesis. Primordial germ cells (PGCs) are the first germ-cell population established during development, and are the common origins of both oocytes and spermatogonia. Unlike in other species, PGCs in birds undergo blood circulation to migrate toward the genital ridge, and are one of the major biological properties of avian PGCs. Germ cells enter meiosis and arrest at prophase I during embryogenesis in females, whereas in males they enter mitotic arrest during embryogenesis and enter meiosis only after birth. In chicken, gonadal sex differentiation occurs as early as embryonic day 6, but meiotic initiation of female germ cells starts from a relatively late stage (embryonic day 15.5). Retinoic acid controls meiotic entry in developing chicken gonads through the expressions of retinaldehyde dehydrogenase 2, a major retinoic acid synthesizing enzyme, and cytochrome P450 family 26, subfamily B member 1, a major retinoic acid-degrading enzyme. The other major biological property of avian PGCs is that they can be propagated in vitro for the long term, and this technique is useful for investigating proliferation mechanisms. The main factor involved in chicken PGC proliferation is fibroblast growth factor 2, which activates the signaling of MEK/ERK and thus promotes the cell cycle and anti-apoptosis. Furthermore, the activation of PI3K/Akt signaling is indispensable for the proliferation and survival of chicken PGCs.

Isolation and Characterization of Chicken Primordial Germ Cells and Their Application in Transgenesis.

Primordial germ cells (PGCs), precursors of functional gametes, offer great promise for the use of genetic resources and transgenesis in chickens. PGCs can be isolated from the developing embryo at diverse early stages and are subsequently expandable in vitro. In vitro proliferating chicken PGCs can facilitate the production of efficient germline chimeras and transgenic chickens. Here, we describe methods to isolate and characterize PGCs from chicken embryos and their application to transgenesis.

Wnt/β-catenin signaling pathway activation is required for proliferation of chicken primordial germ cells in vitro.

Here, we investigated the role of the Wnt/β-catenin signaling pathway in chicken primordial germ cells (PGCs) in vitro. We confirmed the expression of Wnt signaling pathway-related genes and the localization of β-catenin in the nucleus, revealing that this pathway is potentially activated in chicken PGCs. Then, using the single-cell pick-up assay, we examined the proliferative capacity of cultured PGCs in response to Wnt ligands, a β-catenin-mediated Wnt signaling activator (6-bromoindirubin-3′-oxime [BIO]) or inhibitor (JW74), in the presence or absence of basic fibroblast growth factor (bFGF). WNT1, WNT3A, and BIO promoted the proliferation of chicken PGCs similarly to bFGF, whereas JW74 inhibited this proliferation. Meanwhile, such treatments in combination with bFGF did not show a synergistic effect. bFGF treatment could not rescue PGC proliferation in the presence of JW74. In addition, we confirmed the translocation of β-catenin into the nucleus by the addition of bFGF after JW74 treatment. These results indicate that there is signaling crosstalk between FGF and Wnt, and that β-catenin acts on PGC proliferation downstream of bFGF. In conclusion, our study suggests that Wnt signaling enhances the proliferation of chicken PGCs via the stabilization of β-catenin and activation of its downstream genes.

Cryopreservation of chicken primordial germ cells by vitrification and slow freezing: A comparative study

In the present study, we compare a classical slow freezing (SLF) method and an aseptic vitrification (Vitrif) technique to cryopreserve a stable primordial germ cell (PGCs) line issued from the Ardennaise chicken breed. Viability immediately after warming was close to 80% and did not differ between the two cryopreservation methods. Proliferation tended to be slower for both cryopreservation methods compared with controls, but the difference was significant only for Vitrif. No difference was found between the two methods after flow cytometry analysis of stage-specific embryonic antigen-1 expression and reverse transcription–polymerase chain reaction on several factors related to PGC phenotype. After 1 week in culture, all cryopreserved cells reached controls' main morphologic and expanding (viability/proliferation) features. However, SLF generated more unwanted cells clusters than Vitrif. After injection of the PGCs into recipient embryos, vitrified PGCs reported a clear, yet not significant, tendency to colonize the gonad at a higher rate than slow frozen PGCs. SLF in cryovials remains simple, inexpensive, and less technically demanding than Vitrif. Nevertheless, the intrinsic advantages of our aseptic Vitrif method and the present study suggest that this should be considered as safer than classical SLF for cryopreserving chicken PGCs.

Efficient Gene Transfer into Chicken Gonads by Combining Transposons with Polyethylenimine

&lt;p&gt;Transposon mediated transfection is a promising, safe, and convenient way to generate transgenic chicken compared with virus-mediated technology and the in vitro modification of primordial germ cells (PGCs). To establish a simple method for in vivo transfection of chicken PGCs, we applied four different transposon systems (PB, SB, Tol2, and ZB) to investigate the gene transfer efficiency of chicken gonads via direct injection of a mixture of transposon and transposase plasmids and transfection reagent (polyethylenimine, PEI) into the subgerminal cavity of Hamburger and Hamilton stage 2-3 chick embryos. We also compared the effect of the amount of plasmids injected on the gene transfer efficiency of chicken gonads. We found that over 70% of the gonads were green fluorescent protein (GFP)-positive across all four transposon groups, and that the proportion of GFP-positive gonads was not significantly different between different transposons. Some GFP positive cells in gonads were confirmed as germ cells by co-labeling with the germ cell specific antibody. We also found that the proportions of GFP-positive gonads decreased significantly with a decrease of plasmid dose from 100 ng to 20 or 50 ng. Here we revealed that a combination of transposons with PEI is a simple and efficient method for gene transfer into chicken gonads and able to transfect PGCs in vivo that could be used for the production of transgenic chickens.&lt;/p&gt;

Expression of heavy chain-only antibodies can support B-cell development in light chain knockout chickens.

Since the discovery of antibody‐producing B cells in chickens six decades ago, chickens have been a model for B‐cell development in gut‐associated lymphoid tissue species. Here we describe targeting of the immunoglobulin light chain locus by homologous recombination in chicken primordial germ cells (PGCs) and generation of VJCL knockout chickens. In contrast to immunoglobulin heavy chain knockout chickens, which completely lack mature B cells, homozygous light chain knockout (IgL−/−) chickens have a small population of B lineage cells that develop in the bursa and migrate to the periphery. This population of B cells expresses the immunoglobulin heavy chain molecule on the cell surface. Soluble heavy‐chain‐only IgM and IgY proteins of reduced molecular weight were detectable in plasma in 4‐week‐old IgL−/− chickens, and antigen‐specific IgM and IgY heavy chain proteins were produced in response to immunization. Circulating heavy‐chain‐only IgM showed a deletion of the CH1 domain of the constant region enabling the immunoglobulin heavy chain to be secreted in the absence of the light chain. Our data suggest that the heavy chain by itself is enough to support all the important steps in B‐cell development in a gut‐associated lymphoid tissue species.

Improvement of Chicken Primordial Germ Cell Maintenance In Vitro by Blockade of the Aryl Hydrocarbon Receptor Endogenous Activity.

Primordial germ cells (PGCs) are the undifferentiated progenitors of gametes. Germline competent PGCs can be developed as a cell-based system for genetic modification in chickens, which provides a valuable tool for transgenic technology with both research and industrial applications. This implies manipulation of PGCs, which, in recent years, encouraged a lot of research focused on the study of PGCs and the way of improving their culture. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that besides mediating toxic responses to environmental contaminants plays pivotal physiological roles in various biological processes. Since a novel compound that acts as an antagonist of this receptor has been reported to promote expansion of hematopoietic stem cells, we conducted the present study with the aim of determining whether addition of an established AHR antagonist to the standard culture medium used nowadays for in vitro chicken PGCs culture improves ex vivo expansion. We have found that addition of α-naphthoflavone in culture medium promotes the amplification of undifferentiated cells and that this effect is exerted by the blockade of AHR action. Our results constitute the first report of the successful use of a readily available AHR antagonist to improve avian PGCs expansion, and they further extend the knowledge of the effects of AHR modulation in undifferentiated cells.