Chicken Primordial Germ Cells Research Articles

An improved protocol for stable and efficient culturing of chicken primordial germ cells using small-molecule inhibitors.

At present, the most reliable method for creating genetically modified chickens is the modification of the DNA sequence of primordial germ cells (PGCs). However, during embryogenesis, only a small number of chicken PGCs can be obtained. Therefore, in vitro PGC culturing is necessary to obtain sufficient cells for further genetic engineering. Previously reported PGC culturing methods lack versatility. We report here a new protocol for stable and efficient culturing of chicken PGCs using small-molecule inhibitors. The growth rate of PGCs was investigated following the addition of three small-molecule inhibitors, including blebbistatin, into the culture medium. Chicken PGC survival and proliferation rates increased after the addition of small-molecule inhibitors, compared with the untreated control. Blebbistatin was shown to be the most effective inducer of PGC growth. Long-term culturing of PGCs with blebbistatin maintained the morphology of typical PGCs, and these cells expressed marker proteins such as chicken vasa homolog (CVH) and NANOG. Additionally, PGCs transfected with a fluorescent protein gene were shown to migrate into the gonads of the recipient embryo, and progeny derived from PGCs cultured by this method were efficiently obtained. These results demonstrate that small-molecule inhibitors represent a useful tool for stable and efficient chicken PGC culturing.

BMP4 activates the Wnt-Lin28A-Blimp1-Wnt pathway to promote primordial germ cell formation via altering H3K4me2.

ABSTRACTThe unique developmental characteristics of chicken primordial germ cells (PGCs) enable them to be used in recovery of endangered bird species, gene editing and the generation of transgenic birds, but the limited number of PGCs greatly limits their application. Studies have shown that the formation of mammalian PGCs is induced by BMP4 signal, but the mechanism underlying chicken PGC formation has not been determined. Here, we confirmed that Wnt signaling activated via BMP4 activates transcription of Lin28A by inducing β-catenin to compete with LSD1 for binding to TCF7L2, causing LSD1 to dissociate from the Lin28A promoter and enhancing H3K4me2 methylation in this region. Lin28A promotes PGC formation by inhibiting gga-let7a-3p maturation to initiate Blimp1 expression. Interestingly, expression of Blimp1 helped sustain Wnt5A expression by preventing LSD1 binding to the Wnt5A promoter. We thus elucidated a positive feedback pathway involving Wnt–Lin28A–Blimp1–Wnt that ensures PGC formation. In summary, our data provide new insight into the development of PGCs in chickens.

Targeting lentiviral vectors to primordial germ cells (PGCs): An efficient strategy for generating transgenic chickens.

Recent advances in avian transgenic studies highlight the possibility of utilizing lentiviral vectors as tools to generate transgenic chickens. However, low rates of gonadal chimerism and germ line transmission efficiency still limit the broad usage of this method in creating transgenic chickens. In this study, we implemented a simple strategy using modified lentiviral vectors targeted to chicken primordial germ cells (PGCs) to generate transgenic chickens. The lentiviral vectors were pseudotyped with a modified Sindbis virus envelope protein (termed M168) and conjugated with an antibody specific to PGC membrane proteins. We demonstrated that these optimized M168-pseudotyped lentiviral vectors conjugated with SSEA4 antibodies successfully targeted transduction of PGCs in vitro and in vivo. Compared with the control, 50.0%–66.7% of chicken embryos expressed green fluorescent protein (GFP) in gonads transduced by the M168-pseudotyped lentivirus. This improved the targeted transduction efficiency by 30.0%–46.7%. Efficient chimerism of exogenous genes was also observed. This targeting technology could improve the efficiency of germ line transmission and provide greater opportunities for transgenic poultry studies.

A novel F‐box domain containing cyclin F like gene is required for maintaining the genome stability and survival of chicken primordial germ cells

The stability and survival of germ cells are controlled by the germline-specific genes, however, such genes are less known in the avian species. Using a microarray-based the National Center for Biotechnology Information Gene Expression Omnibus dataset, we found an unigene (Gga.9721) that upregulated in the chicken primordial germ cells (PGCs). The unigene showed 97% identities with an uncharacterized chicken cyclin F like gene. The predicted chicken cyclin F like gene was further characterized through expression and regulation in the chicken PGCs. The sequence analysis revealed that the gene shows identities with cyclin F gene and contains an F-box domain. The expression of chicken cyclin F like was detected specifically in the gonads, PGCs, and germline cells. The knockdown of cyclin F like gene resulted in DNA damage and apoptosis in the PGCs. The genes related to stemness and germness were downregulated, whereas, genes related to apoptosis and DNA damage response were increased in the PGCs after the knockdown of chicken cyclin F like. We further observed that the Nanog homeobox controlled the transcriptional activity of chicken cyclin F like gene in PGCs. Collectively, the chicken cyclin F like gene, which is not reported in any other species, is required for maintaining the genome stability of germ cells.

HMEJ-mediated efficient site-specific gene integration in chicken cells

BackgroundThe production of transgenic chicken cells holds great promise for several diverse areas, including developmental biology and biomedical research. To this end, site-specific gene integration has been an attractive strategy for generating transgenic chicken cell lines and has been successfully adopted for inserting desired genes and regulating specific gene expression patterns. However, optimization of this method is essential for improving the efficiency of genome modification in this species.ResultsHere we compare gene knock-in methods based on homology-independent targeted integration (HITI), homology-directed repair (HDR) and homology mediated end joining (HMEJ) coupled with a clustered regularly interspaced short palindromic repeat associated protein 9 (CRISPR/Cas9) gene editing system in chicken DF-1 cells and primordial germ cells (PGCs). HMEJ was found to be a robust and efficient method for gene knock-in in chicken PGCs. Using this method, we successfully labeled the germ cell specific gene DAZL and the pluripotency-related gene Pou5f3 in chicken PGCs through the insertion of a fluorescent protein in the frame at the 3′ end of the gene, allowing us to track cell migration in the embryonic gonad. HMEJ strategy was also successfully used in Ovalbumin, which accounts for more than 60% of proteins in chicken eggs, suggested its good promise for the mass production of protein with pharmaceutical importance using the chicken oviduct system.ConclusionsTaken together, these results demonstrate that HMEJ efficiently mediates site-specific gene integration in chicken PGCs, which holds great potential for the biopharmaceutical engineering of chicken cells.

Investigation of the Guinea fowl and domestic fowl hybrids as potential surrogate hosts for avian cryopreservation programmes

In the last decade, avian gene preservation research has focused on the use of the early precursors of the reproductive cells, the primordial germ cells (PGCs). This is because avian PGCs have a unique migration route through the vascular system which offers easy accessibility. Furthermore, culturing of the cells in vitro, freezing/thawing, reintegration into a recipient embryo and the development of the germ cells can be carried out in well-defined laboratory circumstances. The efficient recovery of the donor genotype and the frequency of germline transmission from the surrogate host animals are still areas which need further development. Thus, the aim of the present study was to investigate an infertile interspecific hybrid (recipient) as an appropriate host for primordial germ cells from native poultry breeds. Guinea fowl × chicken hybrids were produced, the crossing was repeated inversely. The phenotype, the hatching time, the hatching rate, the sex ratio, the presence of own germ cells, the fertility and the phenotype of viable hybrids and the incidence of chromosomal abnormalities of dead hybrid embryos were described. 6.65% viable offspring was obtained with crossing of Guinea fowl females with domestic fowl males. Crossing of domestic fowl hens with Guinea fowl male resulted in lower fertility, 0.14% viable offspring. Based on the investigations, the observed offspring from the successful crossing were sterile male hybrids, thus an extreme form of Haldane’s rule was manifested. The sterile hybrid male embryos were tested by injecting fluorescently labeled chicken PGCs. The integration rate of labeled PGCs was measured in 7.5-day, 14.5-day and 18.5-day old embryonic gonads. 50%, 5.3% and 2.4% of the injected hybrid embryos survived and 40%, 5.3% and 2.4% of the examined gonads contained fluorescent labeled donor PGCs. Therefore, these sterile hybrid males may be suitable recipients for male PGCs and possibly for female PGCs although with lower efficiency. This research work shows that the sterility of hybrids can be used in gene conservation to be a universal host for PGCs of different avian species.

GSK-3 signaling is involved in proliferation of chicken primordial germ cells

Primordial germ cells (PGCs) are precursors of sperms and oocytes and responsible for passing the genetic information from one generation to the next. Chicken PGCs segregate from somatic cells in early embryo and could be isolated and cultured in vitro, making it a useful tool to produce genetically modified animals. However, the number of PGCs isolated from embryo is limited and these cells are not efficient to proliferation in vitro. GSK-3 plays an important role in multiple intracellular signaling pathways and inhibition of GSK-3-mediated β-catenin phosphorylation is known to reduce apoptosis and promote proliferation in T cells and embryo stem cells (ESC). In this study, we investigate the effect of GSK-3 inhibitor on the proliferation of PGCs in vitro and found significant increases of cell proliferation in the culture supplemented with CHIR. We further found that CHIR regulates PGC cell cycle by activating Wnt signaling and antagonizing the apoptosis of PGCs by inhibition of the expression of caspase-3 and Beclin-1. PGCs treated with CHIR expressed the germ cell-related markers and retain the capability to colonize the embryonic gonad after re-introduction to vasculature of HH stage-15 embryos. These results suggest that GSK-3 is involved in cell renewal and apoptosis in chicken PGCs.

PRDM14 and BLIMP1 control the development of chicken primordial germ cells

The differentiation of primordial germ cells (PGCs) is a fundamental step in development. PR domain-containing protein 14 (PRDM14) and B lymphocyte-induced maturation protein 1 (BLIMP1) play pivotal roles in mouse PGC specification. In the present study, we assessed the roles of chicken orthologs of PRDM14 and BLIMP1 in PGC development. PRDM14 and BLIMP1 were expressed in blastodermal cells and PGCs. The in vivo knockdown of PRDM14 or BLIMP1 by introducing a replication-competent retroviral vector expressing shRNAs to the blastodermal stage of embryos reduced the number of SSEA-1 or chicken vasa homologue-positive PGCs on day 5.5–6.5. Since the inhibition of Activin receptor-like kinase 4/5/7 in cultured PGCs reduced the expression of PRDM14, BLIMP1, and NANOG, and that of MEK inhibited PRDM14 expression, the expression of these genes seems to be controlled by Activin A and FGF2 signaling. Overall, PRDM14, BLIMP1, and NANOG seem to be involved in the self-renewal of PGCs in cultured PGCs and embryos.

Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) have facilitated the production of genome-edited animals for use as models. Because of their unique developmental system, avian species offer many advantages as model vertebrates. Here, we report the development of novel chicken models using the CRISPR/Cas9-mediated nonhomologous end joining repair pathway in chicken primordial germ cells (PGCs). Through the introduction of a donor plasmid containing short guide RNA recognition sequences and CRISPR/Cas9 plasmids into chicken PGCs, exogenous genes of donor plasmids were precisely inserted into target loci, and production of transgenic chickens was accomplished through subsequent transplantation of the Z chromosome-targeted PGCs. Using this method, we successfully accomplished the targeted gene insertion to the chicken sex Z chromosome without detected off-target effects. The genome-modified chickens robustly expressed green fluorescent protein from the Z chromosome, which could then be used for easy sex identification during embryogenesis. Our results suggest that this powerful genome-editing method could be used to develop many chicken models and should significantly expand the application of genome-modified avians.-Lee, H. J., Yoon, J. W., Jung, K. M., Kim, Y. M., Park, J. S., Lee, K. Y., Park, K. J., Hwang, Y. S., Park, Y. H., Rengaraj, D., Han, J. Y. Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development.

In vitro culture and characterization of duck primordial germ cells

ABSTRACTThis study aimed to isolate, culture, and characterize duck primordial germ cells (PGCs) and to compare these cells with chicken PGCs. We first cultured Muscovy duck (Cairina moschata) circulating PGCs and gonadal PGCs (gPGCs) in the modified serum-containing medium used to amplify chicken PGCs. gPGCs were found to proliferate better in serum-free chemically defined medium than in serum-containing medium. Thereafter, gPGCs were similarly isolated from 2 other duck breeds, the Pekin duck (Anas platyrhynchos) and the hybrid mule duck (C. moschata × A. platyrhynchos), and amplified for a limited period of time in the chemically defined culture condition, but sufficiently to be characterized and transplanted. Cultured gPGCs of all 3 duck breeds were characterized by Periodic acid-Schiff staining, immunocytochemical staining, and expression analysis of germline-specific and pluripotency genes. Cultured duck gPGCs colonized the gonads after being genetically labeled and injected into recipient embryos. Taken together, these results demonstrate that duck PGCs retain their germline characteristics after being isolated, expanded in vitro, and genetically modified. Further studies are required to establish the optimal conditions for long-term culture of duck PGCs, which may involve supplementing the culture medium with other growth factors or compounds.

Aldehyde dehydrogenase in fresh primordial germ cells as a marker of cell 'stemness'.

SummaryChicken primordial germ cells (PGCs) are the primary pluripotent stem cell types that will differentiate towards germ cells. High aldehyde dehydrogenase (ALDH) activity is considered as a functional marker for the detection of cell 'stemness'. In our study the ALDEFLUOR™ kit was used for determination of ALDH activity in PGCs. PGCs were co-stained with diethylaminobenzaldehyde (DEAB) and ALDH and analyzed by flow cytometry. Our results showed a small cell population (8.0 ± 3.3%) upon preincubation of the cells with the specific inhibitor DEAB, however cells without inhibitor staining showed a fluorescence shift as an ALDH-positive population (70.5 ± 1.6%). These findings indicate higher expression of ALDH in PGCs and ALDH activity can therefore be used as a new functional marker for the detection of cell 'stemness' in chicken PGCs. These results may have importance for characterization of PGCs as a potential genetic resource in poultry. Further research is necessary to elucidate the role of this functional marker in these cells.

Chicken Mesenchymal Stem Cells as Feeder Cells Facilitate the Cultivation of Primordial Germ Cells from Circulating Blood and Gonadal Ridge

Long-term maintenance of chicken primordial germ cells (PGCs) in vitro has tremendous potential for transgenic chicken production. Feeder cells are essential for the establishment and culture of chicken PGCs in vitro. Buffalo rat liver (BRL) cells are the most commonly used feeder cells for PGCs culture; however, this feeder layers from other animal species usually cause immunogenic contaminations, compromising the potential of PGCs in applications. Therefore, we tested chicken source mensenchymal stem cell (MSCs) derived from bone marrow as feeder cells to further improve PGC culture conditions. MSCs derived from chicken bone marrow have a powerful capacity to proliferate and secrete cytokines. We found chicken primordial germ cells derived from circulating blood (cPGCs) and gonads (gPGCs) can be maintained and proliferated with MSCs feeder layer cells. PGCs co-cultured on MSCs feeder retained their pluripotency, expressed PGCs specific genes and stemness markers, and maintained undifferentiated state. Our study indicated that the xeno-free MSCs-feeders culture system is a good candidate for growth and expansion of PGCs as the stepping stone for transgenic chicken research.

Distinct roles of retinoic acid and BMP4 pathways in the formation of chicken primordial germ cells and spermatogonial stem cells.

This study demonstrated different effects of bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) signaling on the induction of germ cell formation in chickens. In vitro, BMP4 significantly promoted primordial germ cell (PGC) formation, while RA promoted spermatogonial stem cell (SSC) formation. Hematoxylin-Eosin (HE) staining of reproductive ridge and testicular slices showed that BMP4 signaling was activated during PGC formation but was inhibited during PGC differentiation into SSC. In contrast, RA signaling was significantly activated during PGC differentiation to SSC. Mechanistically, elevated expression of phosphorylated mothers against decapentaplegic homolog 5 (p-Smad5) activated BMP4 signaling, while inhibition of p-Smad5 significantly reduced the PGC formation. Additionally, BMP4 regulated the PGC formation through histone acetylation and DNA methylation in deleted in azoospermia-like (DAZL) gene. Luciferase report showed RA binding to RARα regulated stimulated by RA 8 (Stra8) promoter activity during SSC formation, while mutations in RAR binding sites inhibited the Stra8 expression and SSC formation. Further, both HAT and HDAC regulated the RARα isoform, and HAT binding to RARα activated the Stra8 transcription. RNA-seq of embryonic stem cells (ESC), PGC, and SSC showed inverse expression of genes related to the BMP4 and RA pathways during PGC and SSC formation. Additionally, Smad5 and Smurf were critical for the interactions between the two pathways. Specifically, through Smurf promotion of Smad5 ubiquitination, RA could inhibit the BMP4 signal transduction. In conclusion, the BMP4 and RA signaling pathways play opposing roles in germ cell formation, driven by epigenetic processes such as phosphorylation, ubiquitination, and histone acetylation.

High fidelity CRISPR/Cas9 increases precise monoallelic and biallelic editing events in primordial germ cells

Primordial germ cells (PGCs), the embryonic precursors of the sperm and egg, are used for the introduction of genetic modifications into avian genome. Introduction of small defined sequences using genome editing has not been demonstrated in bird species. Here, we compared oligonucleotide-mediated HDR using wild type SpCas9 (SpCas9-WT) and high fidelity SpCas9-HF1 in PGCs and show that many loci in chicken PGCs can be precise edited using donors containing CRISPR/Cas9-blocking mutations positioned in the protospacer adjacent motif (PAM). However, targeting was more efficient using SpCas9-HF1 when mutations were introduced only into the gRNA target sequence. We subsequently employed an eGFP-to-BFP conversion assay, to directly compare HDR mediated by SpCas9-WT and SpCas9-HF1 and discovered that SpCas9-HF1 increases HDR while reducing INDEL formation. Furthermore, SpCas9-HF1 increases the frequency of single allele editing in comparison to SpCas9-WT. We used SpCas9-HF1 to demonstrate the introduction of monoallelic and biallelic point mutations into the FGF20 gene and generate clonal populations of edited PGCs with defined homozygous and heterozygous genotypes. Our results demonstrate the use of oligonucleotide donors and high fidelity CRISPR/Cas9 variants to perform precise genome editing with high efficiency in PGCs.

Effects of ten-eleven translocation 1 (Tet1) on DNA methylation and gene expression in chicken primordial germ cells.

Ten-eleven translocation 1 (Tet1) is involved in DNA demethylation in primordial germ cells (PGCs); however, the precise regulatory mechanism remains unclear. In the present study the dynamics of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing PGCs and the role of Tet1 in PGC demethylation were analysed. Results show that 5mC levels dropped significantly after embryonic Day 4 (E4) and 5hmC levels increased reaching a peak at E5-E5.5. Interestingly, TET1 protein was highly expressed during E5 to E5.5, which showed a consistent trend with 5hmC. The expression of pluripotency-associated genes (Nanog, PouV and SRY-box 2 (Sox2)) and germ cell-specific genes (caveolin 1 (Cav1), piwi-like RNA-mediated gene silencing 1 (Piwi1) and deleted in azoospermia-like (Dazl)) was upregulated after E5, whereas the expression of genes from the DNA methyltransferase family was decreased. Moreover, the Dazl gene was highly methylated in early PGCs and then gradually hypomethylated. Knockdown of Tet1 showed impaired survival and proliferation of PGCs, as well as increased 5mC levels and reduced 5hmC levels. Further analysis showed that knockdown of Tet1 led to elevated DNA methylation levels of Dazl and downregulated gene expression including Dazl. Thus, this study reveals the dynamic epigenetic reprogramming of chicken PGCs invivo and the molecular mechanism of Tet1 in regulating genomic DNA demethylation and hypomethylation of Dazl during PGC development.

Three-dimensional culture of chicken primordial germ cells (cPGCs) in defined media containing the functional polymer FP003.

Scalable production of avian cell lines exhibits a valuable potential on therapeutic application by producing recombinant proteins and as the substrate for virus growth due to the special glycosylation occurs in avian species. Chicken primordial germ cells (cPGCs), a germinal pluripotent avian cell type, present the ability of self-renewal, an anchorage-independent cell growth and the ability to be genetically modified. This cell type could be an interesting bioreactor system for industrial purposes. This study sought to establish an expandable culture system with defined components for three-dimensional (3D) culture of cPGCs. cPGCs were cultured in medium supplemented with the functional polymer FP003. Viscoelasticity was low in this medium but cPGCs did not sediment in culture and efficiencies of space and nutrient utilization were thus enhanced and consequently their expansion was improved. The total number of cPGCs increased by 17-fold after 1 week of culture in 3D-FAot medium, an aseric defined medium containing FP003 polymer, FGF2 and Activin A as growth factors and Ovotransferrin as protein. Moreover, cPGC cell lines stably expressed the germline-specific reporter VASA:tdTOMATO, as well as other markers of cPGCs, for more than 1 month upon culture in 3D-FAot medium, indicating that the characteristics of these cells are maintained. In summary, this novel 3D culture system can be used to efficiently expand cPGCs in suspension without mechanical stirring, which is available for long-term culture and no loss of cellular properties was found. This system provides a platform for large-scale culture of cPGCs.

Derivation of chicken primordial germ cells using an indirect Co-culture system

Primordial germ cells (PGCs) are promising genetic resources for avian studies including modified animals. However, chicken PGCs are slow to proliferate and gradually lose germline competency after long-term culture, which hinders their application in avian biotechnology. Thus, we developed a robust method for the isolation and rapid propagation of PGCs using an indirect co-culture system. PGCs derived from a pair of embryonic chicken gonads were expanded to 1 × 106 within 2 weeks, and no sex bias was observed in. These PGCs presented high capacity of germline transmission and produced donor-derived offspring after injection into the chicken embryos. This system allows the efficient gene-banking of chicken species and can facilitate the production of chickens bearing a desired phenotype via genomic editing.

Role of Epigenetic Regulation by the REST/CoREST/HDAC Corepressor Complex of Moderate NANOG Expression in Chicken Primordial Germ Cells.

Primordial germ cells (PGCs), the precursors of gametes, have regulatory mechanisms involving transcription factors and epigenetic modifications. The transcription factor NANOG is a key regulator of germ cell and embryonic stem cell characteristics. However, the epigenetic regulation of NANOG with a histone deacetylase (HDAC) complex in PGCs has not been studied. In this study, we investigated the epigenetic regulation, and in particular the histone acetylation, of NANOG in chicken PGCs. Intriguingly, although NANOG was highly activated in chicken PGCs, the upstream region of its promoter was moderately suppressed by histone deacetylation. HDAC inhibition induced histone H3 lysine 9 acetylation (H3K9ac) and derepressed NANOG expression. Furthermore, knockdown studies revealed that HDAC complex members, such as RE1-silencing transcription factor (REST) and REST corepressor 3 (RCOR3), are important epigenetic modulators of NANOG expression in chicken PGCs. We demonstrate that moderate regulation of NANOG by the REST/CoREST/HDAC complex might be crucial for maintaining the integrity of PGCs.

Disruption of G0/G1 switch gene 2 ( G0S2) reduced abdominal fat deposition and altered fatty acid composition in chicken.

Chicken as a food source is one of the most widespread domestic animals, and it has been used extensively as a research model. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system is the most efficient and reliable tool for precise genome-targeted modification and has generated considerable excitement for industrial applications, as well as biologic science. Unlike in mammals, germline-transmittable primordial germ cells (PGCs) in chicken were used as an alternative strategy for the production of genetically altered chickens. Here, by combining the CRISPR-Cas9 platform and germ cell-mediated germline transmission, we generated G0/G1 switch gene 2 ( G0S2) knockout (KO) chickens, and G0S2 null KO chickens showed a dramatic reduction of abdominal fat deposition without affecting other economic traits. Additionally, G0S2 null KO chickens had altered fatty acid compositions in their blood and abdominal fat compared with wild-type chickens under normal dietary conditions. The global mRNA sequencing data showed that G0S2 disruption in chickens would activate the adipose tissue-specific peroxisomal oxidation pathway, and enoyl-coenzyme A (CoA), hydratase/3-hydroxyacyl CoA dehydrogenase might be a target molecule in metabolic homeostasis in the chicken adipose tissue. Our results demonstrate that the CRISPR-Cas9 system with chicken PGCs can facilitate the production of specific genome-edited chickens for practical applications, as well as basic research.-Park, T. S., Park, J., Lee, J. H., Park, J.-W., Park, B.-C. Disruption of G0/G1 switch gene 2 ( G0S2) reduced abdominal fat deposition and altered fatty acid composition in chicken.

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.