• Korean Journal of Ichthyology
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• v.6 no.2
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• pp.237-243
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• 1994

This work was conducted to study sex differentiation in the black sea bream, Acanthopagrus schlegeli (Bleeker), using a histological method for the appearance of primordial germ cell, formation of primitive gonads, differentiation of female and male from newly hatched larva to the ovotestis stage of fish. The 3~4 primordial germ cells of $6.8{\sim}7.2\;{\mu}m$ in size, which were buried under fibrous mesenchymal tissue between gut duct and notochord of pre-larva with a total length (T.L.) of 2.4 mm at 3 days after hatching. The proto-gonial cells were located in the epithelium of the coelom attached with pigment cells of juvenile with 6.4 mm in T.L. at 21 days after hatching. In juvenile of 20.8 mm in T.L. at 59 days after hatching, the proto-gonial cells were migrated to the retro-peritoneum through the lineshaped primitive gonad composed of fibrous mesenchymal tissue. In juvenile of 7.8 em in T.L. at 186 days after hatching, the mitotic division of proto-gonial cell appeared in the lineshaped primitive gonad having many eosinophilic granule cells and abundant fibrous connective tissue. In juvenile of 9.5 em in T.L. at 254 days after hatching, the gonad was occupied by abundant fibrous connective tissue, bundles of spermatocyte and spermatid. In juvenile of 10.5 cm in T.L. at 13 months after hatching, the gonad was divided into cortical layer and medullary layer. The former was composed of bundles of a few spermatocytes and proto-gonial cells, the latter was filled with the fibrous mesenchymal tissue and a few proto-gonial cells. In juvenile of 14.7 em in T.L. at 16 months after hatching, the gonad was separated into ovarian part and testicular part by the fibrous connective tissue. The ovarian part is consisted of ovarian cavity and oocytes of perinucleolus stage. The testicular part was occupied by spermatogonia in the cyst.

• Development and Reproduction
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• v.12 no.3
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• pp.215-220
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• 2008

Sex differentiation process of the spotted Black Bullhead, Pseudobagrus koreanus, was investigated using fish samples of different age after hatching. The primordial germ cells appeared separately under air bladder in 1-day larva (total length: $6.63{\sim}6.95\;mm$). The primordial gonad with a genital ridge developed in 5-day prelarva ($7.50{\sim}9.36\;mm$). The ovarian differentiation started in about 25-day juvenile ($11.58{\sim}13.21\;mm$). The somatic tissues enlongated in the tip of one end of undifferentiated gonad and fused each other. Thus a small ovarian cavity appeared. The testicular differentiation was initiated in 30-day juvenile ($12.19{\sim}13.72\;mm$). The rudiment of sperm duct was appeared in the lower part of the undifferentiated gonad. In 50-day juvenile ($16.28{\sim}17.06\;mm$), the ovary started to fill with peri-nucleolus oocytes, and the spermatogonia started to develop. In 250-day juvenile ($35.49{\sim}51.12\;mm$), the ovary became bigger and filled with oocytes, and the number of spermatogonia started to increase. Considering these results, the spotted Black Bullhead could be a differentiated type in sex differentiation and gonochorism in sexuality.

• 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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• 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.

• Korean Journal of Poultry Science
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• v.40 no.3
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• pp.163-169
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• 2013

Cryopreservation of poultry semen has been reported, but preservation of female genetic material has not been possible because of the unique anatomical and physiological characteristics of the avian egg. Thus an alternative strategy for conservation of oviparous species of animals must be developed. Recent technological developments for producing germline chimeras by the transfer of primordial germ cells (PGCs) into recipient embryos has enabled the conservation and retrieval of chicken genetic resources in their complete form. In the present study, fertilized eggs were incubated for about 5.5 days to obtain embryos at stage 28. The whole embryo was collected from the germinal gonad using a fine glass micro pipette under a microscope. The PGCs were then purified using MACS method. Two commercially available cryoprotectants (A and B) were used to preserve the PGCs, and EG were used as a control. The average recovery rate of PGCs after thawing was 35.5% and 60.5% with the A and B treatments, respectively. There was no significant difference between B treatments and control, which showed an average recovery rate of 52.8%. However, the recovery rate obtained using A cryoprotectant (35.5%) was significantly lower than using treatment control and B. The average viability of the PGCs after thawing were 77.9% and 77.4% for cryoprotectants A and B, respectively, and the control were was 81.6%. There was no statistically significant difference between the two treatments and control. It was concluded that all of the available cryoprotectants examined in this study could be used for preservation of PGCs from embryos. Further experiments to produce germline chimera from PGCs preserved using this techniques are strongly recommended.

• The Korean Journal of Zoology
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• v.33 no.4
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• pp.435-445
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• 1990

Gonadotropin releasing horrnone (GnRH) is known to be extrahypothalamically localized with a broad range including gonad. It remains, however, unknown whether GnRH is locally synthesized in the gonad. The present srudy aims to identity expression and cellular localization of GnRH-Iike mRNA and immunoreactive GnRH in the rat gonad. GnRH radioimmunoassay and chromatographic extracts on G-50 sephadex column showed that rat gonadal extracts contained a substantial amount of immunoreactive GnRH similar to the hypothalamic and synthetic GnRH. Although a wide distribution of immunostainable GnRH-like molecule with different cell types in the rat ovary was observed, the major cell population hybridized with GnRH probe appears to be granulosa. theca cells and corpus luteum. Immunoreactive GnRH-Iike peptides were distributed m various regions of testis, including spermatogenic cells, Sertoli cells and Leydig cells. In situ hybridization revealed that positive signals of GnRH-Iike mRNA were predominandy present in Sertoli cells within some seminiferous tubules, but absent in the outside of seminiferous tubules in the testis. This study clearly demonstrated that GnRH-Iike molecule present in the rat gonad may be resulted from the local synthetic machinery of GnRH supporting the notion that this peptide may act as autocrine and/or paracrine role in intra-gonadal communication.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2001.08a
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• pp.36-37
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• 2001

큰가리비는 자웅이체이다. 난환형성과정은 난모세포의 발달정도에 다라 다르게 나타나고 있다. 난자형성과정은 난원세포기, 전난황형성난모세포기, 초기난황형성난모세포기, 후기난황형성난모세포기, 성숙난모세포기의 연속적인 5단계의 과정으로 나눌 수 있었다. 전난황형성기 난모세포질 내에서는 핵주변 구역에 골지장치와 수많은 공포들 및 미토콘드리아들이 출현하고 있는데 이들은 차후, 지방적 형성에 관여한다. 난황형성전기난모세포(previtellogenic oocyte)에서는 지방적 및 지질과립들이 핵막 근처에서 출현하여 피질층쪽으로 분산되는 반면, 같은 발달 단계의 난모세포질의 피질구역에서는 피질과립들(단백질성 난황과립)이 처음으로 생성되어 난황막 근처의 피질층에서 핵주변 구역쪽으로 분산.분포된다. 난황형성후기 난모세포에서는 세포질 내의 골지장치, 공포, 미토콘드리아, 그리고 조면소포체들이 자율합성에 의해 난황과립 형성에 관여하고 있다. 반면 외인성 물질들인 지질형태의 과립들, 다량의 글리코겐 입자들이 생식상피 내에서 출현하고 있는데. 이들 물질이 생식상피에서 난황막 구조물인 미세융모를 통해 난황형성 후기 난모세포의 날질 내로 통과해 들어가는 현상이 관찰되었다. 이와같은 현상은 난황형성이 일어날 때에 heterosynthesis가 일어나고 있음을 시사한다. 완숙난모세포의 난경은 약 50~60$\mu\textrm{m}$이다. 정자형성과정은 정원세포기, 제1차정모세포기, 제2차정모세포기, 정세포기, 정자기의 연속적인 5단계로 나눌수 있었다. 정셍포기에서 정자로 변태되는 과정 중에 침체의 분화과정이 있는데 이에는 1. Golgi phase, 2. Cap phase 3. acrosome phase, 4. maturation phase의 단계를 거쳐 첨체가 완성된다. 정자는 원시적 형태를 이루고 있으며 4개의 미토콘드리아가 부핵을 형성하고 있다. 완숙정자 두부의 길이는 대략 $3 \mu$m 이며, 미부의 길이는 약 $30 \mu$m정도이다. 정자 미부편모의 axoneme은 중앙의 2개의 미세소관(microtubule)과 주변에 위치한 9개의 2중 미세소구관(microtublue)으로 이루어져 있다.

• 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.

• Development and Reproduction
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• v.4 no.2
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• pp.195-201
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• 2000

Sex differentiation process of the spotted sea bass, Lateolabrax maculatus, was investigated by histological method. The fish samples were collected from just after hatching to 365 days later. The primordial germ cells and genital ridge were appeared separately hanging under air bladder in 30-day larva (total length: 11.7~13.2 mm), and were unified into the undifferentiated gonads in 40-day larva (12.5~14.0 mm). The ovarian differentiation was started in 60-day juvenile (23.6~27.0 mm). The somatic tissues were elongated in tip of both ends of undifferentiated gonad and were fused each other. The complete ovarian cavity was appeared in 80-days juvenile(33.1~42.5 mm). The testicular differentiation was initiated in 70-day juvenile (24.8~31.6 mm). The rudiment of sperm duct was appeared in the center of the undifferentiated gonad. The meiosis of germ cells in the ovary was started in 168-day juvenile (88.0~115.4 mm). In 287-day juvenile (175.1~233.6 mm), the ovary was filled with both of chromatin stage and perinucleolus stage oocytes. The meiosis of male germ cells was started in 245-day juvenile (124.4~168.3 mm). However, the seminiferous tubules of testis were filled with numerous sperm in 365-day juvenile (162.5~253.8 mm). The sex ratio of male and female was 1:1.38. Considering these results, the spotted sea bass was showed differentiated type in sex differentiation and gonochorism in sexuality.

• Korean Journal of Animal Reproduction
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• v.12 no.1
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• pp.11-14
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• 1988

Utilizatin of primordial germ cell(PGC) as transferor of genetic materials is great potential in manipulating genes to promote genetic performances in chicken. This study explored that PGCs from early embryos as vehicle for molecular breeding strategles were isolated, these chromosomally marked donor PGCs were transplanted to germinal crescent of host embryos, and genetic materials of donor PGC were identified at the proliferative stage in host gonads.