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. 2025 Aug;104(8):105308.
doi: 10.1016/j.psj.2025.105308. Epub 2025 May 17.

Characterization of primordial germ cells from EG&K stage X chicken embryos

Dongyang Chen  1 , Yifei Zhi  1 , Xiaoxuan Jia  1 , Na Ji  1 , Jiale Zhang  1 , Jiongming Liang  1 , Mingxia Ran  1 , Xingting Liu  1 , Huiyan Xu  1 , Yangqing Lu  2
Affiliations

Characterization of primordial germ cells from EG&K stage X chicken embryos

Dongyang Chen et al. Poult Sci. 2025 Aug.

Abstract

Primordial germ cells (PGCs) derived from Eyal-Giladi and Kochav (EG&K) stage X embryos (XPGCs) represent the earliest germ cells in poultry. However, little is known about the XPGCs due the difficulty in the isolation and propagation of these cells. In this study, we report a method for the stable isolation and efficient in vitro propagation of XPGCs. We conducted a thorough analysis of the characteristics exhibited by XPGCs. First, we identified an average of 42 PGCs at this stage by using an DAZL-2a-mCherry genetically modified chickens. Three stable cell lines, one male and two female, were established from eight isolates using a filter paper ring and differential adhesion. Germ cell-related protein markers, including DAZL, CVH, and SSEA-1, were detected in the established XPGCs. Re-introduction of the XPGCs into the blastoderms of EG&K stage X embryos or the cardiovascular systems of Hamilton and Hamburger (HH) stage 14 embryos showed that the XPGCs retained the ability to migrate to the gonads after long-term culture. XPGCs demonstrated a lower growth rate in vitro but a higher potential of migrating to the gonads than PGCs derived from the embryonic gonads of HH stage 27-31. The ability to isolate, culture, and characterize XPGCs advances our knowledge of early-stage germ cell development and provides a valuable cell tool for genetic conservation and genome editing in chickens.

Keywords: EG&K stage X embryo; In vitro culture; Primordial germ cell; chicken.

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Conflict of interest statement

Declaration of competing interest We would like to submit the enclosed manuscript entitled “Characterization of primordial germ cells from EG&K stage X chicken embryos” to be considered for publication in Poultry Science. No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. All the authors have approved the manuscript that is enclosed. We deeply appreciate your consideration of our manuscript, and we look forward to receiving comments from the reviewers. If you have any queries, please don’t hesitate to contact me at the address below.

Figures

Fig 1
Fig. 1
Isolation and culture of XPGCs. (A) A schematic of the isolation and culture of the XPGCs. (B) A fluorescence image of an EG&K stage X blastoderm from a DAZL-mCherry chicken embryo. (C) A fluorescence image of the mixed cell population from the blastoderm. (D) Images of cells obtained every 6 days during cell line establishment. (E) Cell proliferation curve of XPGCs. (F) Sex identification of XPGC cell lines using PCR. Scale bar: 500 μm (B), 100 μm and 20 μm (C), and 100 μm (D).
Fig 2
Fig. 2
Characterization of the XPGCs cultured in vitro. (A) Alkaline phosphatase testing of XPGCs. (B) RT-PCR to detect the germline and stem cell-related gene expression of PouV, Nanog, Dazl, Cvh, and Cdh in XPGCs. (C) Immunofluorescence staining for the germ stem cell markers CVH, DAZL, and SSEA1 in XPGCs. Scale bar: 100 μm and 50 μm (A), and 20 μm (C).
Fig 3
Fig. 3
A comparison of the proliferation potential between XPGCs and gPGCs. (A) A total of 1 × 104 cells each of XPGCs and gPGCs were cultured under identical conditions for 3 days, after which the number of cells was determined. (B) Expression of the cell proliferation-related genes Pcna and Ccnd1 in XPGCs and gPGCs. (C) Analysis of the cell cycle distribution of XPGCs and gPGCs using flowing cytometry. *P < 0.05.
Fig 4
Fig. 4
Migration ability of XPGCs and gPGCs. (A) Schematic of the XPGC transplantation into the subgerminal cavity of the blastoderm of EG&K stage X host embryos. (B) Fluorescence image of the embryonic gonads 6.5–7 days after injecting 500 XPGCs. (C) Schematic of XPGC transplantation into the blood circulation system of HH stage 13–16 host embryos. (D) Fluorescence image of the embryonic gonads 5 days after injecting 1 × 104 XPGCs. (E) Fluorescence image of the embryonic gonads after transplantation of XPGCs and gPGCs. (F) The cell count of mCherry-positive PGCs in the embryonic gonads after transplantation of XPGCs and GPGCs. (G) Expression of the cell migration-related gene Cxcr4 in XPGCs and gPGCs. (H) Scanning electron micrograph of XPGCs and gPGCs. *P < 0.05 and **P < 0.01. Scale bar: 500 μm (B, D and E), 5 μm (H).
Fig 5
Fig. 5
Germ cell-related gene expression levels of XPGCs and gPGCs. *P < 0.05 and **P < 0.01.
Fig 6
Fig. 6
Cryopreservation and germline transmission of XPGCs. (A) Proliferation of XPGCs after cryopreservation and thawing. (B) Cell proliferation curve of XPGCs after cryopreservation and thawing. (C) G0 chimeric chickens. (D) Fluorescence images of the testes and ovaries of G0 chimeric chickens. (E) PCR analysis of semen from adult G0 chimeric roosters. (F) Fluorescence image of G1 chicken embryos. Scale bar: 50 μm (A), 1 mm (D), and 500 μm (F).
Fig 7
Fig. 7
Germline genetic efficiency of XPGC. (A) The strategy for determining the XPGC germline transmission efficiency. (B) Three distinct genotypes of G1 offspring derived from chimeric chicken: homozygous White Leghorn chick (-/-, left), heterozygotes (±, middle), and homozygous Donglan chick (+/+, right). (C) Frequency of germline transmission from founder chimeras to G1 chickens of the three genotypes.
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