• Proceedings of the Korea Society of Poultry Science Conference
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• 2005.11a
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• pp.66-67
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• 2005

The domestic chicken (Gallus gallus) is an important model for research in developmental biology because its embryonic development occurs in ovo. To examine the mechanism of embryonic germ cell development, we constructed proteome map of gonadal primordial germ cells (gPGC) from chicken embryonic gonads. Embryonic gonads were collected from 500 embryos at 6 day of incubation, and the gPGC were cultured in vitro until colony formed. After 7-10 days in cultured gPGC colonies were separated from gonadal stroma cells (GSCs). Soluble extracts of cultured gPGCs were then fractionated by two-dimensional gel electrophoresis (pH 4-7). A number of protein spots, including those that displayed significant expression levels, were then identified by use of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and LC-MS/MS. Of the 89 gPGC spots examined, 50 yielded mass spectra that matched avian proteins found in on-line databases. Proteome map of thistype will serve as an important reference for germ cell biology and transgenic research.

• Journal of Animal Science and Technology
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• v.55 no.5
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• pp.427-434
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• 2013

This study was conducted to establish methods for preserving chicken primordial germ cells (PGCs) for long-term storage in liquid nitrogen and for developmental engineering or preservation of species. The purpose of this study is to clarify the effects of fetal bovine serum (FBS) or chicken serum (CS) treatment on the viability of cryopreserved PGCs from Korean Native Chicken (Ogye). PGCs separated from a germinal gonad of an early embryo at day 5.5-6 (stage 28) were suspended in a freezing medium containing freezing and protective agents (dimethyl sulfoxide (DMSO), ethylene glycol (EG) and glycerol). The values from 0, 5, 10, and 15 % DMSO plus FBS treatment were 21.6, 30.36, 36.42, 50.39, and 48.36 %, respectively. The viability of PGCs after freeze-thawing was significantly higher for 10% EG plus FBS treatment than for 10% EG + FCS treatment (p<0.05) (64.36% vs. 50.66%). This study establishes a method for preserving chicken PGC that enables systematic storage and labeling of cryopreserved PGC in liquid nitrogen at a germplasm repository and an ease of entry into a database. In the future, the importance for this new technology is that poultry lines can be conserved while work is being conducted to improve the production of germline chimeras.

• Journal of Aquaculture
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• v.16 no.4
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• pp.240-244
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• 2003

The primitive gonad development in Paralichthys olivaceus was classified into (ⅰ) primordial germ cell (PGC), (ⅱ) genital ridge type (GRT), (ⅲ) primitive gonad type I (PGT I) and (ⅳ) primitive gonad type II stages. In the control group, PGC was recognizable on the 3rd days after hatching, and the primodial gonad after 24th days, while it was around the 22nd day after hatching in the group exposed to PCBs 3.0 $\mu\textrm{g}$/L for 30 days. Likewise, the progress of kidney development was recognized in four stages; unitubular type of mesonephric duct (UTMD), the branched mesonephric duct (BMD), convoluted tubule formation (CTF) and glomerulus appearing (GA) stage. It was structurally completed between the 25th and 30th day after hatching in two groups. There was no significant difference (p>0.05) in the time scale of development of gonad, and kidney between control and the PCBs - exposed group.

• Reproductive and Developmental Biology
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• v.31 no.2
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• pp.127-131
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• 2007

Embryonic germ (EG) cells are undifferentiated stern cells isolated from cultured primordial germ cells (PGC). These cells share many characteristics with embryonic stem cells including morphology and pluripotency. Undifferentiated porcine EG cell lines demonstrating capacities of differentiation both in vitro and in vivo have been established. Since EG cells can be cultured indefinitely in an undifferentiated state, whereas somatic cells in primary culture are often unstable and have limited lifespan, EG cells may provide inexhaustible source of karyoplasts in nuclear transfer (NT). In this study the efficiencies of NT using porcine EG and fetal fibroblast cells were compared. Two different techniques were used to perform NT. With conventional NT procedure (Roslin method) involving fusion of donor cells with enucleated oocytes, the rates of development to the blastocyst stage in EG and somatic cell NT were 16.8% (59/351) and 14.5% (98/677), respectively. In piezo-driven microinjection (Honolulu method) of donor nuclei into enucleated oocytes, the rates of blastocyst formation in EG and somatic cell NT were 11.9% (15/126) and 9.4% (9/96), respectively. Regardless of NT methods used in this study, EG cell NT gave rise to comparable rate of blastocyst development to somatic cell NT. Overall, EG cells can be used as karyoplast donor in NT procedure, and embryos can be produced by EG cell NT that may be used as an alternative to conventional somatic cell NT.

• Proceedings of the Korea Society of Poultry Science Conference
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• 2004.11a
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• pp.9-10
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• 2004

We developed a new panel of markers for the characterization of chicken PGCs. The results of immunostaining demonstrated that anti-SSEA-3, anti-SSEA-4, anti-integrin 6, and anti-integrin 1 antibodies. and STA and DBA bound specifically to chicken PGCs. These reagents could be used to characterize chicken PGCs together with conventional marker reagents such as PAS and anti-SSEA-1 antibody. We also showed that double staining of PGCs with the newly developed markers was feasible, which might contribute to rapid detection and accurate characterization of chicken PGCs.

• Journal of Embryo Transfer
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• v.9 no.3
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• pp.255-260
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• 1994

원시생식세포(primordial germ cell; PGC)는 성성숙 이후에 기능을 갖는 생식세포의 근원이 되는 세포로서, 다능성을 갖고 있는 것으로 알려져 있다. 그러므로 chimera 및 유전자 변환동물 생산을 위해 널리 사용되어 온 배아주(embrynic stem; ES)세포를 대신할 다른 세포계라고 생각되어져 많은 연구가 진행되고 있다. 본 실험은 체외배양을 통하여 원시생식세포의 증식과 확립을 위해 배양조건을 구명하고, 또한 성장인자의 효과를 검증하기 위하여 실시되었다. 원시생식세포는 12.5일째의 ICR 생쥐태아의 원시생식선 융기조직으로부터 추출하였으며, DMEM + 20% FCS + nucleosides + antibiotics로 조성된 sDMEM 배양액을 사용하여 mitomycin C로 전처리한 되먹임세포단층(feeder layer)위에서 체외배양하였다. bFGF 및 LIF를 20, 40ng/ml농도로 각각 또는 함께 첨가하여 성장인자의 효과를 검토하였다. 원시생식세포는 성에 따라 유의적인 colony 형성율을 보였고(♂:1.9 colonies / genital ridge, ♀:1.3 colonies / genital ridge), bFGF 및 LIF의 첨가 및 첨가농도에 따라서도 유의성 있는 결과를 보였다(0.3~1.9 colonies / genital riege). 그러나 3회 이상 계대배양을 할 경우, 원시생식세포의 colony를 4% prarformaldehyde로 20분간 고정한 후, tris-maleate buffer(pH 9.0)로 10분간 3회 세정하였다. Fast Red로 염색을 실시한 결과, 대부분의 colony가 염색반응을 보여 다능성을 갖는 원시생식세포의 colony임이 입증되었다. 그러나 대부분의 colony가 3회 이상의 계대배양시 생종율이 급격히 떨어지는 것을 감안하면, 또 다른 미지의 성장인자나 보다 적절한 배양조건이 요구된다고 생각된다.

• Korean Journal of Poultry Science
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• v.40 no.3
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• pp.197-205
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• 2013

This study established a method for preserving chicken primordial germ cells (PGCs) that enables long-term storage in liquid nitrogen ($LN_2$) for preservation of the species. The purpose of this study was to compare the effects of Ethylene Glycol (EG) and Propylene Glycol (PG) on viability of cryopreserved PGCs with vitrification in Korean Native Chicken (Ogye), and to fine should be find or to the optimal protocol for PGCs freezing. One of the important components of cryopreservation process is cryopreservation medium that plays a vital role in preventing cellular injury during freeze-thawing. Cryoprotective agents have been known to improve cell viability after freeze-thawing. PGCs obtained from the germinal gonade of 5.5~6 day (stage 28) chick embryos, using the MACS method were suspended in a freezing medium containing a freezing and protecting agents. Gonads were harvested from stage 28 chick embryos and pooled in groups of 10E embryos, contributing gonads to the cell suspension. The gonadal cells, including PGCs, were then frozen in 1 of the following cryoprotectant treatments: 2.5% EG, 5% EG, 10% EG, 2.5% PG, 5% PG, 10% PG, and 0% cryoprotectant as a control. Effects of exposure to vitrification solution and vitrification, with different concentrations of the cryoprotectant solution, were examined. After freezing and thawing, survival rates of the frozen-thawed PGCs from the 0, 2.5, 5, 10 and 15% EG plus FBS treatment were 44.24%, 64.51%, 85.63%, 80.51% and 73.52% (p<0.05), respectively. The viability of PGCs after freeze-thawing was significantly higher for 10% EG plus FBS treatment than for 10% PG + FBS treatment (p<0.05)(85.63% vs 66.81%). Therefore, these systems may contribute in the improvement of cryopreservation for a scarce species in birds preservation. This study established a method for preserving chicken PGC that enables systematic storage and labeling of cryopreserved PGCs in liquid N at a germplasm repository and ease of entry into a database. In the future, the importance for this new technology is that poultry lines can be conserved while work is being conducted on improving the production of germline chimeras.

• Korean Journal of Ichthyology
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• v.21 no.3
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• pp.141-148
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• 2009

Sex differentiation and gonad development were investigated in a marine medaka species, Oryzias dancena (Beloniformes; Teleostei). The average time to hatch was 11 days post-fertilization (dpf) at $25^{\circ}C$. Primordial germ cell (PGC) was first observed at 5 dpf and migrated to presumptive gonadal area between the gut and pronephric duct at 9 dpf. Male and female gonads were morphologically differentiated at 12 days post-hatching (dph). Early oocytes at perinucleolus stage as well as the formation of spermatid and efferent duct were observed at 28 dph. At 6 weeks of age, the ovary exhibited yolk granulation in many oocytes, while testis possessed a considerable number of spermatogonia and spermatids. The first ovulation was observed in 9-week-old females, and at the same age, males contained fully-matured spermatozoa. Data obtained in this study indicate that the gonad differentiation of O. dancena is the typical type of differentiated gonochorism.