• Proceedings of the Korea Society of Poultry Science Conference
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• 2002.11a
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• pp.98-99
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• 2002

Collection of large number of gonadal Primordial germ cells(gPGCs) is a prerequisite factor for improving germline transmission efficacy in the aves, In this study a magnetic-activated cell sorter(MACS) was applied for improving retrieval efficacy of quail gPGCs and the migration capacity of MACS-separated gPGCs was further examined after being transplanted to recipient embryos. We also induced germline chimeras by transfer of MACS-separated quail gPGCs at the efficiency of 17.4% on average.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2001.11a
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• pp.69-70
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• 2001

Lectins have great potential as to determine the alternation of the distribution of cell surface carbohydrates during cellular development and differentiation. Here, we investigated the presence and distribution of cell surface carbohydrates on chicken primordial germ cells (PGCs) during the migration and gonadal stages using a variety of lectins. A total of six FITC-labelled lectins from several specificity classes were used: ConA (glucose/mannose), WGA (N-acetylglucosamine), STA (N-acetylglucosamine), DBA (N-acetylgalactosamine/galactose), UEA-I (fucose) and PHA-E (oilgosaccharide). As a results, PGC-specific binding was observed in STA. PGCs of migration stage (2.5- and 5.5-day embyos) were STA-positive whereas PGCs of 10-day embryonic gonad were not. The results suggest that N-acetylglucosamine residuse are present specifically in migrating chicken PGCs and changes during development.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2001.11a
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• pp.40-46
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• 2001

The use of pluripotent stem cells has tremendous advantages for various purposes but these cell lines with proven germ-line transmission have been completely established only in the mouse. Embryonic germ (EG) cell lines are also pluripotent and undifferentiated stem cells established from primordial germ cells (PGCs). This study was conducted to establish and characterize the chicken EG cells derived from gonadal primordial germ cells. We isolated gonadal PGCs from 5.5-day-old (stage 28) White leghorn (WL) embryos and established chicken EG cells lines with EG culture medium supplemented with human stem cell factor (hSCF), murine leukemia inhibitory factor (mLIF), bovine basic fibroblast growth factor (bFGF), human interleukin-11 (hIL-11), and human insulin-like growth factor-I (hIGF-I). These cells grew continuously for 4 months (10 passages) on a feeder layer of mitotically active chicken embryonic fibroblasts. These cells were characterized by screening with the Periodic acid-Shiff＇s reaction, anti-SSEA-1 antibody, and a proliferation assay after several passages. As the results, the chicken EG cells maintained characteristics of undifferentiated stem cells as well as that of gonadal PGCs. When cultured in suspension, the chicken EG cells successfully formed an embryoid body and differentiated into a variety of cell types when re-seeded onto culture dish. The chicken EG cells were injected into blastodermal layer at stage X and dorsal aorta of recipient embryo at stage 14 (incubation of 53hrs) and produced chimeric chickens with various differentiated tissues derived from the EG cells. The germline chimeras were also successfully induced by using EG cells. Thus, Chicken EG cells will be useful for the production of transgenic chickena and for studies of germ cell differentiation and genomic imprinting.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.8
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• pp.1188-1191
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• 1999

Primordial germ cells (PGCs) of White Leghorn chicken embryos as a donor were transferred to Rhode Island Red chicken embryos as a recipient. At 48-50 h (stage 13-15) of incubation of fertilized eggs, donor PGCs, which were taken out from blood vessels of donor embryos, were injected into blood vessels of recipient embryos. Sex of the treated embryos was determined after the transfer of PGCs using remaining blood samples. In the present experiments, survival rate of the treated embryos was 33.3% for homo-sexual and 35.4% for hetero-sexual transfers of PGCs, respectively, when determined at 17 days of incubation. In this study, most of the treated embryos could not survive more than 18 days of incubation, though the reason for that was not clarified in the present work. The gonalds removed from embryos that died after 18 days of incubation and the organs from newly hatched chicks were examined for morphological and histological features. The gonads removed from the embryos with homo-sexual transfer of PGCs showed normal development in appearance. On the contrary, some (35.3%) of the embryos with hetero-sexual transfer of PGCs possessed abnormal gonads similar to ovotestis by histological observation. In cases where the gonads developed to be normal organs (64.7%) the sex of embryos was the same as recipient ones. The present results suggest that hetero-sexual transfer of the PGCs may bring about the possibility of development of the embryos bearing sexually different gametes, spermatogonia or oogonia.

• Journal of Animal Science and Technology
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• v.55 no.5
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• pp.417-425
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• 2013

We sought to provide a method for freezing and preserving primordial germ cells, or an avian germ cell of a bird, as a material for developmental engineering or species preservation. The aim of this study was to compare the efficacy of slow freezing with a vitrification method for the cryopreservation of chicken primordial germ cells (PGCs). PGCs obtained from the germinal gonad of day 5.5-6 day (stage 28) cultured chick embryos, using the MACS method, were classified into two groups: slow freezing and vitrification. We examined the viability of PGCs after Cryopreservation. Four freezing methods were compared with each other, including the following: Method 1: The PGCs were frozen by a programmed freezer in a plastic straw, including 2.0 M ethylene glycol (EG) as cryoprotective additive (slow freezing) Method 2: The PGCs were vitrified in a plastic straw, including 8.0 M EG, plus 7% polyvinylpyrrolidone (PVP) (rapid freezing). Method 3: The slow freezing was induced with a cryotube including 2.0 M EG Method 4: The PGCs were frozen in a cryotube including 10% dimethyl suloxide (DMSO) (rapid freezing). After freezing and thawing, survival rates of the frozen-thawed PGCs from Method 1 to 4were 76.4%, 70.6%, 80.5% and 78.1% (p<0.05), respectively. The slow freezing ($-80^{\circ}C$ programmed freezer) method may provide better survival rates of frozen-thawed PGCs than the vitrification method for the cryopreservation of PGCs. Therefore, these systems may contribute to the cryopreservation of a rare avian species.

• Proceedings of the Korea Society of Poultry Science Conference
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• 1999.11a
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• pp.11-33
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• 1999

This study was conducted to determine the embryonic stages for the isolation of the highest number of PGCs and to improve PGCs enrichment method. The primordial germ cells(PGCs) from different sources of chick embryos were isolated. The embryonic stage having the highest number of PGCs from each sources was selected ; 1-day-old embryos for germinal crescent (stage 6-8), 2.5-day-old embryos for blood (stage 17-18) and 5.5-day-old embryos for gonad (stage 27-28). The number of PGCs from one embryonic germinal crescent, blood and gonad was about 87$\pm$1.8, 103$\pm$4.0, and 932$\pm$10.9, respectively. The viability of PGCs after Ficoll from each sources was similar, showing approximately 70%. the PGCs enrichment method was improved using Ficoll density gradient centrifugation. After this step the purity of PGCs from germinal crescent, blood, and gonad was 45$\pm$9.10%, 85$\pm$1.18%, and 86$\pm$0.19%, respectively. Also, PGCs were picked up by mouth pipette to improve the purity. This improved method for the separation of PGCs from different sources will serve as a useful too to preserve the foundation stocks of poultry and to produce germline chimeras.

• Proceedings of the KSAR Conference
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• 2001.10a
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• pp.47-47
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• 2001

One of the problems associated with in vitro culture of primordial germ cells (PGCs) is the large loss of cells during the initial period of culture. This study characterized the initial loss and determined the effectiveness of two classes of apoptosis inhibitors, protease inhibitors and antioxidants, on the ability of porcine PGCs to survive in culture. Results from electron microscopic analysis and in situ DNA fragmentation assay indicated that porcine PGCs rapidly undergo apoptosis when placed in culture. Additionally,？ 2-macroglobulin, a protease inhibitor and cytokine carrier, and N-acetylcysteine, an antioxidant, increased the survival of PGCs in vitro. While other protease inhibitors tested did not affect survival of PGCs, all antioxidants tested improved survival of PGCs (p〈0.05). Further results indicated that the beneficial effect of the antioxidants was critical only during the initial period of culture. Finally, it was determined that in short-term culture, in the absence of feeder layers, antioxidants could partially replace the effect(s) of growth factors and reduce apoptosis. Collectively, these results indicate that the addition of ？2-macroglobulin and antioxidants can increase the number of PGCs in vitro by suppressing apoptosis.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.276-276
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• 2004

Pluripotent stem cells have been generated from two embryonic sources. ES cells are generated from ICM of blastocyst stage embryos, and embryonic germ (EG) cells are generated from primordial germ cells (PGCs). Both ES and EG cells are pluripotent and present important characteristics such as high levels of alkaline phosphatase (AP) activity, multi-cellular colony formation, normal and stable karyotypes, continuously passaging ability, and the capability of differentiation into all three embryonic germ layers. (omitted)

• Korean Journal of Poultry Science
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• v.28 no.1
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• pp.69-76
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• 2001

Avian primordial germ cells (PGCs) originate from the epiblast and appear in the germinal crescent. These PGCs enter the developing blood vessels during stage 10∼12 (H&H), circulate in the blood stream, migrate into the developing gonadal anlage and differentiate into germ cells. However, it is not clear until when the migratory ability of PGC is maintained. This study was conducted to examine whether migratory ability is present in PGCs from the gonad at later embryonic developmental stages. In the present study, gonads were dissected from 5-, 6- and 10-day old quail embryos and treated with trypsin-EDTA. Gonadal PGCs (gPGCs) were purified by Ficoll-density-gradient-centrifugation and labeled with PKH26 fluorescent dye. The PKH26-labeled gPGCs were microinjected into the blood vessel of the recipient quail embryo. Manipulated recipients were incubated for 3 days, embedded in paraffin and sdctioned. The foreign gPGCs were detected by fluorescent and confocal laser microscopy. As a result, quail gPGCs, from 10, 6 and 5 day old embryos could migrate through the recipient blood stream at early stage and settle in the gonads. Thus, results suggest that gPGCs from upto 10-day old embryos keep properties seen in circulating PGC. Therefore, the PGCs of 10-day old embryonic gonads can be used for the tools of genetic manipulation.

• Applied Microscopy
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• v.15 no.1
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• pp.1-12
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• 1985

In this study, the pathway and date of migrating Primordial germ cells (PGCs) were observed light microscopically and ultrastructural changes of them during migration were observed by electron microscopic examination. For these purpose, alkaline phosphatase reactions were used for identifying the PGCs and acid phosphatase reactions were used for observing their degenerating activities. Also, effects of actinomycin D on the migration of PGCs were examined. According to these results, at the 9th gestation day, PGCs were observed in the endodermal cells of yolk sac, at the 11th gestation day, they were seen in the hindgut and then entered into the dorsal mesentery by the 13th gestation day. At the 14th gestation day, they were located in the genital ridges. When PGCs were located in the hindgut and genital ridges, the positive reactions of alkaline phosphatase were dominated, but acid phosphatase reactions were limited in all stage except they were in dorsal mesentery. However, these reactions were lessened in case of actinomycin D treatment. By electron microscopic examination, PGCs had pseudopodia, tail process, trailing cytoplasm and nuage as the ultrastructural characteristics. In addition, these morphological features were damaged by actinomycin D treatment.