Long-term <i>in vitro</i> culture and preliminary establishment of chicken primordial germ cell lines

<div><p>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 <i>Tert</i>, <i>DAZL</i>, <i>POUV</i>, and <i>NANOG</i>. 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.</p></div

Compound culture system for chicken PGCs.

<p><b>(A)</b> Morphology of PGCs in the presence of a feeder layer and at 5 days after bFGF withdrawal (Bar = 100 μm). <b>(B)</b> Cell proliferation rate of PGCs in the presence of a feeder layer and at 5 days after feeder layer withdrawal. <b>(C)</b> The proliferation and viability rates of mitomycin C treated STO cells detected by a CCK-8 assay; STO cells were treated with various concentrations of mitomycin C (10, 15, 20, 30 μg/mL) for 2, 3, and 4 h, when their confluence reached 60%. <b>(D)</b> Growth curve of STO cells treated with different concentrations of mitomycin C. <b>(E)</b> Morphology of STO cells treated with different concentrations of mitomycin C. <b>(F)</b> Cell proliferation rate of PGCs in the presence of BRL conditioned medium (CM) and at 5 days after CM withdrawal. <b>(G)</b> The proliferation and viability rates of PGCs as detected by the CCK-8 assay, the proportion of CM in the medium was 0%, 20%, 40%, 50%, 60%, and 80%, respectively.</p

Long-term cultured PGCs retain their differentiation ability <i>in vitro</i>.

<p><b>(A)</b> Morphological changes in PGCs upon RA induction. The long-term cultured PGCs were treated with 10 μmol/L RA for 8 days and showed differentiation into presumptive SSC-like cells (Bar = 100 μm). <b>(B)</b> Quantitative RT-PCR was performed to verify the related genes in the presence or absence of RA induction for 0, 2, 4, 6, and 8 days, respectively, using SSC-specific (<i>STRA8</i> <b>(a)</b>, <i>SYCP3</i> <b>(b)</b>), germness-related (<i>DAZL</i> <b>(c)</b>), and stemness-related (<i>NANOG</i> <b>(d)</b>) genes. The gene expression values at day 6 are shown in <b>(e)</b>. <b>(C)</b> Immunocytochemistry for SSC marker genes <i>integrinα6</i> (Red) and <i>integrinβ1</i> (Green) at day 8 in the presence or absence of RA induction (Bar = 25 μm).</p

Isolation and culture of chicken PGCs.

<p><b>(A-D)</b> Morphology of cultured PGCs. <b>(A)</b> PGCs (white arrow) were isolated and primarily cultured with gonadal stroma cells (black arrow) and blood cells (red arrow) from chicken embryos at Stage 27 (Bar = 100 μm). <b>(B)</b> PGC colony (white arrow) formation after 3 days of primary culture (Bar = 100 μm). <b>(C)</b> Gonadal stroma cells could not support PGCs (white arrows) after 7 days of primary culture (Bar = 100 μm). <b>(D)</b> PGCs (white arrows) were then subcultured on feeder layers (mitomycin C treated STO cells) (Bar = 25 μm). <b>(E)</b> Morphology of STO cells (Bar = 100 μm). <b>(F)</b> Morphology of BRL cells (Bar = 100 μm).</p

The technical scheme for chicken PGCs long-term culture in vitro and preliminary establishment of cell lines.

<p>The technical scheme for chicken PGCs long-term culture in vitro and preliminary establishment of cell lines.</p

Long-term cultured PGCs maintain high pluripotency and migration ability.

<p><b>(A-D)</b> Immunocytochemical analysis of cultured PGCs. PGCs cultured for 180 days were immunostained with antibodies raised against SSEA-1 <b>(A)</b>, c-kit <b>(B)</b>, cDAZL <b>(C)</b>, and Sox2 <b>(D)</b>. <b>(E)</b> qRT-PCR analysis of <i>POUV</i>, <i>NANOG</i>, and <i>DAZL</i> in cultured PGCs (cultured for 15, 68, 180, and 268 days and in thawed cells) (Bar = 25 μm). <b>(F)</b> Migration of cultured PGCs into the germinal crescent. Approximately 3000 PGCs, cultured for 200 days, were labeled with PKH26 and then transferred into the subgerminal cavities of blastoderm embryos. The cells injected were 268-day cultured PGCs (above) and DMEM (below). Labeled cells (red) were detected in the germinal crescent. (Bar = 5 mm) <b>(G)</b> Gonadal migration of cultured PGCs. Approximately 3000 cells were labeled with PKH26 and then injected into the blood vessels of recipient embryos at Stage 14–17. From the left to right, the cells injected were 268-day cultured PGCs, 15-day PGCs, thawed PGCs, DF-1, and DMEM. Labeled cells (red) were detected in the embryonic gonad (Bar = 1 mm). <b>(H)</b> Cells isolated from the isolated gonad with the PKH26 labeled PGCs (Bar = 100 μm).</p

Discovery of piRNAs Pathway Associated with Early-Stage Spermatogenesis in Chicken

<div><p>Piwi-interacting RNAs (piRNAs) play a key role in spermatogenesis. Here, we describe the piRNAs profiling of primordial germ cells (PGCs), spermatogonial stem cells (SSCs), and the spermatogonium (Sp) during early-stage spermatogenesis in chicken. We obtained 31,361,989 reads from PGCs, 31,757,666 reads from SSCs, and 46,448,327 reads from Sp cells. The length distribution of piRNAs in the three samples showed peaks at 33 nt. The resulting genes were subsequently annotated against the Gene Ontology (GO) database. Five genes (<i>RPL7A</i>, <i>HSPA8</i>, <i>Pum1</i>, <i>CPXM2</i>, and <i>PRKCA</i>) were found to be involved in cellular processes. Interactive pathway analysis (IPA) further revealed three important pathways in early-stage spermatogenesis including the FGF, Wnt, and EGF receptor signaling pathways. The gene <i>Pum1</i> was found to promote germline stem cell proliferation, but it also plays a role in spermatogenesis. In conclusion, we revealed characteristics of piRNAs during early spermatogonial development in chicken and provided the basis for future research.</p></div

RT-qPCR of candidate genes.

<p>Lowercase letters indicated p ≤ 0.01.</p

Venn diagram analysis of common different piRNAs in two groups (PGCs vs. SSCs and SSCs vs. Sp).

<p>Venn diagram analysis of common different piRNAs in two groups (PGCs vs. SSCs and SSCs vs. Sp).</p

Number of piRNAs up- or down-regulated among two groups (PGCs vs. SSCs and SSCs vs. Sp).

<p>The x-axis represents the number of piRNAs, and the y-axis reflects the groups.</p