• 한국발생생물학회지:발생과생식
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• 제1권2호
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• pp.189-202
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• 1997

The hypothalamic peptide GnRH plays a central role in the regulation of the mammalian reproductive axis. Recent studies suggested that GnRH stimulates or inhibits the ovarian steroidogenesis and gametogenesis directly. Our previous report indicated that GnRH gene is expressed in adult rat ovary as well as in hypothalamus and that the expressed GnRH may induce the follicular atresia and apoptosis of ovarian granulosa cells in rat. Therfore, we studied whether GnRH gene is expressed in the mouse fetal ovary, when the germ cells are degenerating by apoptosis during gonad diffeerentiation. Mouse fetal gonads were obtained on the 12, 15,18 and 20th day of gestation from the mother mice superovulated (10 IU PMSG and 10 IU hCG) and mated. The morphological changes of fetal ovaries were examined histochemically by hematoxylin-eosin staining. The fetal sex was confirmed by PCR methods for sexing. RT-PCR methods were used to examine the expression of GnRH gene and the sex steroid hormones were determined by conventional radioimmunoassays. The levels of estradiol (E) and progesterone (P) were increaseduntil 18th day of gestation and then E was decreased just before parturition. The morphological changes of fetal gonadal tissue sections showed the ovarian development and coincided with the result of PCR analysis for sexing using ovary- or testis- specific oligonucleotide primers. Immunoreactive GnRH in placenta was decreased gradually until the end of gestation but fetal brain and ovarian GnRH were increased. The level of GnRH gene expression was increased during fetal ovarian development from 12 till 18th day and decreased suddenly on 20th day just before birth. From these results, it is suggested that ovarian GnRh may play a regulatory role on the germ cell differentiation of fetal ovary.

• 한국가금학회:학술대회논문집
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• 한국가금학회 2000년도 제17차 정기총회 및 학술발표
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• pp.75-77
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• 2000

Retaining migratory activity is a prerequisite for the manipulation and use of PGCs. This study was conducted to examine whether migratory activity is retained in the primordial germ cells(PGCs) from gonads at the later embryonic developmental stage. In the present study, gonads were dissected from 5-, 6- and 10-day-old quail embryos and treated with trypsin-EDTA for the degradation of gonadal tissue. 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 vessels of recipient quail embryo. After further incubation of 3 days, the manipulated recipients were embedded in paraffin and sectioned. The gPGCs were detected by their fluorescence under the fluorescent microscopy and the confocal laser microscopy. As a result, 10-day-old quail gPGCs as well as 5-and 6-day-old gPGCs, could migrate to recipient embryonic gonads and settle down. These results suggest that the 10-day-old gPGCs have the properties of circulating PGCs at early stage. Therefore the PGCs from 10-day old embryonic gonads can be used for the tools of genetic manipulation.

• Animal cells and systems
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• 제10권4호
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• pp.227-231
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• 2006

Micro-deletions at specific loci of the Y chromosome have been observed frequently in male infertility patients, suggesting that genes in these regions are involved in male germ cell development. DAZ is a representative male infertility gene at the AZFc locus of the Y chromosome. Since DAZ contains an RNA binding motif along with so-called a DAZ domain, it was proposed to participate in RNA metabolism during spermatogenesis. A mouse gene homologous to the human DAZ gene has been cloned and named Dazl (DAZlike). Dazl is autosomal and expressed in the testis and also at a low level in the ovary. Male mice homozygous for the Dazl null allele have small testes with a few spermatogonia and almost complete absence of germ cells beyond the spermatogonial stage, suggesting the requirement of Dazl for entry or progression through meiosis. However, its exact cellular functions have not been understood yet. In order to investigate cellular functions of Dazl, we decided to isolate candidate interacting protein genes of the mouse Dazl, using yeast two-hybrid screening. A number of candidate Dazlinteracting proteins have been isolated, such as Bprp, Acf, Hgs, Murr1, Nbak3 and Ranbp9, but dynein light chain 1 (Dlc1) was most predominant. A strong interaction of Dazl with Dlc1 suggests that Dazl might function as an mRNA adaptor to the dynein motor complex.

• Animal cells and systems
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• 제16권2호
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• pp.104-113
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• 2012

The nucleolus is a distinct nuclear territory involved in the compartmentalization of nuclear functions. There is some evidence of a relationship between nuclear fragmentation during spermatogenesis and chromatoid body (CB) formation. The CB is a typical cytoplasmic organelle of haploid germ cells, and is involved in RNA and protein accumulation for later germ-cell differentiation. The goal of this study was to qualitatively and quantitatively describe the nucleolar cycle during the spermatogenesis of $Phrynops$ $geoffroanus$ (Reptilia Testudines), and compare this nucleolar fragmentation with CB formation in this species through the use of cytochemical and ultrastructural analysis. Qualitative analysis showed a fragmentation of the nuclear material after pachytene of the first meiotic division in the primary spermatocytes. Quantitative analysis of the nucleolar cycle revealed a significant difference in the number of nucleoli and in the size of the nucleolus between spermatogonia and early spermatids. Using ultrastructural analysis, we recorded the beginning of the CB formation process in the cytoplasm of primary spermatocytes at the same time as when nuclear fragmentation occurs. In the cytoplasm of primary spermatocytes, the CB was observed in association with mitochondrial aggregates and the Golgi complex. In the cytoplasm of early spermatids, the CB was observed in association with lipid droplets. In conclusion, our data show that the nucleolus plays a role in the CB formation process. During spermatogenesis of $P.$ $geoffroanus$, the CB is involved in some important biological processes, including acrosome formation and mitochondrial migration to the spermatozoon tail and middle piece region.

• 치과방사선
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• 제28권1호
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• pp.87-109
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• 1998

The present study was designed to elucidate the effects of the Co-60 γ irradiation and/or calcium­deficient diet on the dentin and cementum formation of rat molar. The pregnant three-week old Sprague­Dawley rats were used for the study. The experimental group was divided into two groups, irradiation/normal diet group and irradiation/calcium-deficient diet group. The control group was non­irradiation/normal diet group. The abdomen of the rats at the 19th day of pregnancy were irradiated with single absorbed dose of 350cGy. The rat pups were sacrificed on the 14th day after delivery and the maxillae including molar tooth germ were taken. The specimens including the 1st molar tooth germ were prepared to make tissue sections for light and transmission electron microscopy. Some of tissue sections for light microscopy were stained immunohistochemically with anti-fibronectin antibody. The results were as follows; 1. The Hertwig's epithelial root sheath cells, which are related to the differentiation of the tooth-forming cells, showed irregular cellular arrangement, decrease of intercellular junctional complex, and decreased immunoreactivity to the fibronectin after irradiation. These were more severe in the irradiation/calcium-deficient diet group. 2. The cementoblasts at the cementum-forming area showed chromatin clumpings after irradiation. The immu noreactivity to the fibronectin was weaken after irradiation, especially irradiation/calcium-deficient diet group. 3. The odontoblasts at the dentin-forming area showed increase of lysosomes in the cytoplasm and destruction of intercellular junctional complex. The irradiation/calcium-deficient diet group showed decrease of number and density of the electron dense particles and a large number of vacuoles scattered in the dentin matrix. The immunoreactivity was weaken.

• 한국발생생물학회지:발생과생식
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• 제13권4호
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• pp.271-280
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• 2009

태반은 성체줄기세포의 보고이다. 특히 양막상피세포는 배아줄기세포의 줄기세포 능력을 나타내는 세포 표면 표시자들을 그대로 발현하는 줄기세포로 알려져 있다. 하지만 상피세포를 실험실에서 지지세포 없이 대량 증식 배양하는 것은 상피세포가 가지고 있는 내인성 성격으로 인해 어렵다. 본 연구에서는 디티오트레이톨(Dithiothreitol; DTT)과 ROCK 저해제(Rho-associated kinase inhibitor)를 이용하여 양막상피세포를 분리하고 배양하는데 있어서 임상적용이 가능한 수준의 세포를 얻었고, 최적의 세포상태를 유지하였다. 본 연구에서 분리배양된 양막상피세포는 상피세포의 특성과 줄기세포의 특성을 발현하였다. 결론적으로 줄기세포 치료를 이용한 재생의학의 관점에서 인간태반 유래 양막상피줄기세포는 아무런 윤리적인 논란을 일으키지 않는 주요한 줄기세포 치료제의 재료로서 여러 가지 질병 치료에 사용될 수 있을 것이다.

• Journal of Microbiology and Biotechnology
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• 제14권5호
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• pp.891-900
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• 2004

UDP-N-Acetylglucosamine(GlcNAc):$\beta$1,4-D-mannoside$\beta$-l ,4N-acetylglucosaminyltransferase-III (GnT-III) and UDP-N-GlcNAc:$\alpha$-6-D-mannosid$\beta$-1,6N-acetylglucosaminyltransferase-V(GnT - V) activities were determined in human hepatoma cell lines and metastatic colon cancer cells, and their activities were compared with those of normal liver cells and fetal hepatocytes. GnT-III activities were higher than those of GnT-V in hepatic carcinoma cells. When the two enzyme activities were assayed in highly metastatic colon cancer cells, GnT - V activities were much higher than those of GnT-III. When GlcN, GlcN-biant-PA and UDP-GlcNAc were used as substrates, the enzymes displayed different kinetic properties between hepatic and colon cancer cells, depending on their metastatic potentials. Normal cells of two origins had characteristically very low levels of GnT-III and -V activities, whereas hepatoma and colon cancer cells contained high levels of activities. These data were supported by RT-PCR and Northern blot analyses, showing that the expression of GnT-III and -V mRNAs were increased in proportion to the enzymatic activities. The increased GnT-III, md -V activities were also correlated with increased glycosylation of the cellular glycoproteins in hepatoma and colon cancer cells, as examined by lectin blotting analysis by using wheat germ glutinin (WGA), erythroagglutinating phytohemagglutinin (E-PHA), leukoagglutinating phytohemagglutinin (L-PHA), and concanavalin A (Con A). Treatment with retinoic acid, a differentiation agent, resulted in decreases of both GnT-III and -V activities of HepG2 and HepG3 cells. In colon carcinoma cells, however, treatment with retinoic acid resulted in a reduction of GnT-V activity, but not with GnT-III activity. Although the mechanism underlying the induction of these mzymes is unclear, oligosaccharides in many glycoproteins have been observed of cancer cells.

• BMB Reports
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• 제51권9호
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• pp.437-443
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• 2018

Non-homologous end joining (NHEJ), and to a lesser extent, the error-free pathway known as homology-directed repair (HDR) are cellular mechanisms for recovery from double-strand DNA breaks (DSB) induced by RNA-guided programmable nuclease CRISPR/Cas. Since NHEJ is equivalent to using a duck tape to stick two pieces of metals together, the outcome of this repair mechanism is prone to error. Any out-of-frame mutations or premature stop codons resulting from NHEJ repair mechanism are extremely handy for loss-of-function studies. Substitution of a mutation on the genome with the correct exogenous repair DNA requires coordination via an error-free HDR, for targeted transgenesis. However, several practical limitations exist in harnessing the potential of HDR to replace a faulty mutation for therapeutic purposes in all cell types and more so in somatic cells. In germ cells after the DSB, copying occurs from the homologous chromosome, which increases the chances of incorporation of exogenous DNA with some degree of homology into the genome compared with somatic cells where copying from the identical sister chromatid is always preferred. This review summarizes several strategies that have been implemented to increase the frequency of HDR with a focus on somatic cells. It also highlights the limitations of this technology in gene therapy and suggests specific solutions to circumvent those barriers.

• 대한한의학방제학회지
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• 제12권2호
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• pp.155-162
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• 2004

The purpose of this study is to examine the protective effect of Cynomorii herba on $H_2O_2$-induced cytotoxicity in male germ cells. The effects were studied by a modified MTT assay. Hydrogen peroxide significantly induced cytotoxicity in GC-1 spg cells, and co-treatment or pre-treatment of Cynomorii herba extract has reduced the cytotoxicity in a dose dependant manner. These results suggest that Cynomorii herba extract increases the survival rate of GC-1 spg cells through the antioxidant effect against $H_2O_2$-induced cytotoxicity.

• 대한의생명과학회지
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• 제19권1호
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• pp.32-40
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• 2013

Pluripotent stem cells (PSCs) have enormous potential in the biomedical sciences because they can grow continuously and differentiate into any kind of cell in the body. However, for future application in regenerative medicine, it is still a challenge to control the differentiation of PSCs without using genetic materials. To control the differentiation of PSCs, small molecules might be the best substitute for genetic materials considering the following advantages: small size, which enables penetration of plasma membrane; easy-to-modify structure; and low chance of genetic recombination in treated cells. Herein, we introduce small molecules that induce the neuroectodermal differentiation of mouse embryonic stem cells (ESCs). The small molecules were identified via ESC-based consecutive screenings of small-molecule libraries composed of 324 natural compounds or 93 selected drugs. The natural compounds discovered in the first screening were used to select 93 structurally similar drugs out of 1,200 approved drugs. In the second screening, among the 93 compounds, we found 4 drugs that induced the neuroectodermal differentiation of ESCs. These drugs were progesteroneor corticoid-derivatives. Our results suggest that small molecules targeting the progesterone receptor or glucocorticoid receptor could be used as chemical tools to induce the differentiation of PSCs into a specific germ lineage.