• Clinical and Experimental Reproductive Medicine
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• 제33권3호
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• pp.189-198
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• 2006

목적: 본 연구진은 난자성숙 과정의 조절 기작을 규명하기 위하여 생쥐의 미성숙 난자와 성숙난자에서 차이 나게 발현하는 유전자의 목록을 얻은 바 있다. 이들 유전자 중에서 Bcl-2 homolog인 Diva 유전자가 난자에 특이적으로 발현함을 본 연구를 통해 규명하였는데 이러한 Diva의 기능을 밝혀내기 위하여 immunoprecipitation (IP)과 Mass Spectrometry (MS)를 이용하여 Diva와 결합하여 상호작용하는 단백질을 동정하고자 하였다. 연구방법: NIH/3T3 세포주에 Diva를 encoding하는 pCMV-FLAG-Diva를 24 시간 동안 과발현 시키고 대조군으로는 유전자 없는 pCMV-FLAG empty vector를 transfection 하였다. FLAG에 특이적인 항체로 IP하여 Diva와 결합하는 면역복합체를 형성하게 한 후 이를 12% SDS-polyacrylamide gel 상에서 전기 영동하였고 Coomassie Blue 염색을 통해 단백질 발현양상을 관찰하였다. 대조군에서는 관찰되지 않으면서 Diva 유전자가 발현하는 실험군에서만 확인되는 밴드를 오려내어 trypsin을 사용하여 in-gel digestion 한 후 MS 분석을 시행하였다. 모든 mass spectra는 4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA)에 의해 positive reflector mode에서 얻어졌다. 이렇게 얻어진 단백질들은 MASCOT Peptide Mass Fingerprint software (Matrixscience, London)을 이용하여 NCBI nonredundant database를 찾아서 동정하였다. 결과: Diva를 과발현하는 세포주에서만 관찰되는 15개 밴드에 대한 MS/MS 분석 결과, Diva와 결합하는 단백질로서 actin과 그 외에 ${\alpha}$-actinin, tropomyosin, tropomodulin 3 등의 actin-binding 단백질을 동정하였다. Diva를 과발현하는 NIH/3T3 세포주에서 면역 복합체를 형성하는 actin과 tropomyosin이 실제 난소 조직에서도 Diva와 결합하는지 IP와 Western blot을 통해 확인한 결과, actin과 tropomyosin 모두 Diva와 결합함을 확인함으로써, Diva는 이 두 단백질과 난소에서 상호작용함을 알 수 있었다. 결론: 본 연구는 Diva와 결합하여 상호작용하는 단백질들이 cytoskeletal system의 actin filament와 관계 있음을 규명한 최초의 보고이다. Diva가 actin과 tropomyosin과 결합하는 것을 고려해볼때, 난자 특이적인 Diva는 아마도 난자성숙 동안에 cytoskeletal system 을 조절하는 역할을 할 것으로 사료된다.

• Animal cells and systems
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• 제5권1호
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• pp.51-57
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• 2001

We have previously shown that oocyte maturation is induced by an immobilized progesterone, progesterone-3-carboxymethyloxime - bovine serum albumin conjugate (P-BSA) in Rana dybowskii. In this study, we confirmed the maturation inducing activity of P-BSA on Xenopus oocyte and examined the binding character of the immobilized progesterone on the surface of Xenopus oocytes after removal of the vitelline layer. P-BSA induced maturation of Xenopus oocytes but E-BSA failed to do so as observed in Rana. Binding of the immobilized progesterone, fluorescein isothiocyanate-labeled progesterone-3-0-carboxymethyloxime-BSA (P-BSA-FITC) on the devitellined oocytes surface was examined by fluorescence confocal microscopy. The binding affinity of P-BSA-FITC to the devitellined oocyte was higher than that of estrogen-BSA-FITC (E-BSA-FITC) or testosterone-BSA-FITC (T-BSA-FITC). The binding disappeared in the presence of excess free progesterone but not in the presence of free estrogen. Maximum binding occurred after two-hours of incubation with P-BSA-FITC at pH 7.5. Stronger binding occurred in oocytes at stage Vl than stage IV, and in vitro treatment of hCG enhanced the binding. Taken together, these results suggest that a specific receptor for progesterone exists on the plasma membrane of Xenopus oocytes and that progesterone acts initially on this putative receptors and triggers generation of membrane-mediated second messengers during the early stage of oocyte maturation In amphibians.

• Asian-Australasian Journal of Animal Sciences
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• 제23권1호
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• pp.17-24
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• 2010

In the antral follicle phase, several layers of cumulus cells surround the oocyte and play an important support and regulation role in oocyte development and maturation via intercellular communications and interactions between oocytes and cumulus cells. However, information on stage specific gene expression in swine during the phase is not well understood. To investigate the function of cumulus cells during in vitro maturation of porcine oocytes and gene expression, suppression subtractive hybridization (SSH) was performed to screen genes that were differentially expressed between cumulus-oocyte complexes (COCs) and naked oocytes (NOs). Utilizing mRNAs from in vitro maturation oocytes, a SSH cDNA library from COCs as the tester and NOs as the driver was constructed. The SSH cDNA library was then screened using dot blot analysis. Results showed that a total of 70 clones randomly selected from the library were differentially expressed. Among these, 41 exhibited high homology to known genes and 11 were novel expressed sequences tags (ESTs). Four differentially expressed genes, including bfgf, sprouty 2, egr and btc, were further studied by real time quantitative PCR; results confirmed an increased expression of respective mRNA in COCs compared with NOs, which suggests that these factors may play an important role in oocyte development and maturation.

• 한국발생생물학회지:발생과생식
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• 제17권1호
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• pp.63-72
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• 2013

A specific inhibitor of RNA polymerase II, ${\alpha}$-amanitin is broadly used to block transcriptional activities in cells. Previous studies showed that ${\alpha}$-amanitin affects in vitro maturation of cumulus-oocyte-complex (COC). In this study, we evaluated the target of ${\alpha}$-amanitin, and whether it affects oocytes or cumulus cells (CCs), or both. We treated ${\alpha}$-amanitin with different time period during in vitro culture of denuded oocytes (DOs) or COCs in comparison, and observed the changes in morphology and maturation status. Although DOs did not show any change in morphology and maturation rates with ${\alpha}$-amanitin treatment, oocytes from COCs were arrested at metaphase I (MI) stage and CCs were more scattered than control groups. To discover causes of meiotic arrest and scattering of CCs, we focused on changes of cumulus expansion, gap junctions, and cellular metabolism which to be the important factors for the successful in vitro maturation of COCs. Expression of genes for cumulus expansion markers (Ptx3, Has2, and Tnfaip6) and gap junctional proteins (Gja1, Gja4, and Gjc1) decreased in ${\alpha}$-amanitin-treated CCs. However, these changes were not observed in oocytes. In addition, expression of genes related to metabolism (Prps1, Rpe, Rpia, Taldo1, and Tkt) decreased in ${\alpha}$-amanitin-treated CCs but not in oocytes. Therefore, we concluded that the transcriptional activities of CCs for supporting suitable transcripts, especially for its metabolic activities and formation of gap junctions among CCs as well as with oocytes, are important for oocytes maturation in COCs.

• Clinical and Experimental Reproductive Medicine
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• 제21권3호
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• pp.285-296
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• 1994

Follicular oocytes were released from the graafian follicles of ovaries from 3-4 weeks old mice. The spontaenous maturation of these follicular oocyes was inhibited by the treatment of dbcAMP and progesterone and these oocytes were cultured for 2-8hr in the Modified Hank's balanced salt solution(MHBS). Ethylenediaminetetraaceticacid(EDTA) and calmoudulin antagonist, trifluoperazine (TFP) were treated to the culture medium in order to investigate whether these chemical agents inhibit calcium uptake into the oocyte and oocyte maturation. $^{45}Ca^{2+}$, 10-${\mu}$Ci/ml was added to the culture medium during the culture period. $^{45}Ca^{2+}$uptake into the oocytes was examined whether and when various kind of oocyte maturation inhibiting agents inhibit or stimulate the influx of calcium into oocytes. Dibutyryl cAMP and progesterone decrease $^{45}Ca^{2+}$uptake into the oocytes and synergistic inhibiting effect of dbcAMP and progesterone was prominent at much lower dosages. Calcium uptake into oocytes seems to be higher during first 2 hour culture period rather than next 4hr culture. After 8hr culture, calcium uptake level of the oocytes which GVBD already took place gradually approached to the level of those which were maintained at GV by the treatment of dbcAMP and progesterone. However, $^{45}Ca^{2+}$uptake into the GV maintained oocytes did not change at all even after 8hr culture period. In addition, calcium chelating agent, EDTA inhibited calcium uptake into oocytes as well as nuclear maturation of oocytes. Lower dosage used in the present study did not inhibit calcium uptake as well as oocyte maturation.

• Reproductive and Developmental Biology
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• 제28권3호
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• pp.187-190
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• 2004

PKC는 그들의 cofactor-requirments에 따라 cPKC, nPKC 그리고 aPKC, 3그룹으로 나어진다. 마우스 난 성숙과정에 있어서 cPKC 및 nPKC의 activators인 PMA의 영향에 대한 많은 결과가 보고되었다. 그러나 각각의 그룹에 대한 차별화된 영향에 대하여는 밝혀져 있지 않다. Mezerein의 analog인 thymeleatoxin은 cPKC의 특이적인 activator로 보고되어져 있다. 본 연구에서는 specific cPKC activator인 thymeleatoxin의 마우스 난 성숙과정에의 영향을 제1감수분열 재개 능(germinal vesicle break down, GVBD)과 제1 극체 형성 능(1st polar body extrusion)을 조사하여 cPKC및 nPKC activator인 PMA와 비교 검토하였다. 그 결과 GVBD IC50는 thymeleatoxin에서 ～400nM, PMA에서는 ～50nM이었으며, 제1극체 방출의 IC50는 thymeleatoxin에서 ～200nM, PMA에서는 ～20nM이었다. 이들 결과는 Thymeleatoxin의 GVBD나 1st polar body extrusion 저해효과가 PMA에 비하여 1/8～1/10인 것으로 나타났다. 이들 결과는 GVBD나 제1극체 형성을 포함하는 난 성숙과정에서 cPKC보다 상대적으로 nPKC의 관여가 깊음을 보여 준다.

• 한국어류학회지
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• 제22권1호
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• pp.56-60
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• 2010

기름종개와 왕종개로부터 기원한 단성생식집단인 Cobitis hankugensis-Iksookimia longicorpa complex는 대부분 암컷으로만 구성되어 있으며 최근에 1종류의 2배체와 2종류의 3배체집단이 확인되었다. 이들의 난막구조를 조사한 결과 3가지 종류로 구분될 수 있었다. 융모형은 이들의 부모종인 왕종개에서 확인되었으며, 과립형은 기름종개와 3배체집단, 그리고 과립을 가진 융모형은 2배체집단에서 나타났다. 특히 과립형을 가지는 3집단에서는 그들의 길이와 밀도에서 서로 차이를 보였다. 이러한 구조들은 cobitid complex에서 처음 보고될 뿐 아니라 2배체, 3배체 complex집단을 분류하는 데 이용될 수 있다.

• Reproductive and Developmental Biology
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• 제33권2호
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• pp.93-98
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• 2009

In this study, bovine Dnmt1 cDNA was sequenced and detected Dnmt1 mRNA level in bovine tissues by northern blot, methylation pattern of genome by southern blot, specific localization of Dnmt1 in mouse and bovine preimplantation embryos by immunocytostaining and Dnmt1 protein level in ovary and testis by western blot. Bovine Dnmt1 cDNA sequence showed more homology with that of human than mouse and rat. The RNA level of Dnmt1 was 10 times higher expression in placenta than other tissues. This indicates that placenta was hypermethylated compared to others organs. The genomic DNA could not be cut by a specific restriction enzyme (HpaII) in placenta, lung and liver of bovine. It suggests that Dnmt1 in some somatic cells was already methylated. Dnmt1, which has the antibody epitope 1316~1616, was distributed in nucleus and cytoplasm including the stage of pronuclear stage and maturation of oocyte and gradually weaken to blastocyst stage compare to negative. In addition, Dnmt1 was strongly expressed in tetraploid embryo and cloned 8-cell than IVF 8-cell. An aberrant pattern of DNA methylation in cloned embryo may be abnormal development of fetus, embryonic lethality and placenta dysfunction. The somatic specific band (190kDa) was appeared in ovary and testis, but oocyte specific band (175kDa) was not. Further investigations are necessary to understand the complex links between the methyltransferases and the transcriptional activity of genes in the cloned bovine tissues.

• The Korean Journal of Physiology and Pharmacology
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• 제5권2호
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• pp.157-163
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• 2001

The effects of dimethyl sulfoxide (DMSO) were studied in two groups of Xenopus oocytes, one expressing ATP sensitive $K^+\;(K_{ATP})$ channel comprised of sulfonylurea receptor SUR1 and inwardly rectifying $K^+$ channel subunit Kir6.2, and the other expressing renal $K_{ATP}$ channel ROMK2. At concentrations of $0.3{\sim}10%$ (vol/vol) DMSO inhibited whole cell Kir6.2/SUR1 currents elicited by bath application of sodium azide (3 mM) in a concentration-dependent manner. The inhibition constant and Hill coefficient were 2.93% and 1.62, respectively. ROMK2 currents, however, was not affected significantly by DMSO. The results support the idea that DMSO inhibits $K_{ATP}$ channel expressed in Xenopus oocyte through a protein-specific mechanism(s) that remains to be further elucidated.

• Clinical and Experimental Reproductive Medicine
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• 제39권4호
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• pp.127-131
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• 2012

Primordial follicles are formed prenatally in mammalian ovaries, and at birth they are fated to be activated to primary follicles, to be dormant, or to die. During the early stage of folliclulogenesis, the oocyte undergoes dynamic alterations in expression of numerous genes, which are regulated by transcription factors. Several germ-cell specific transcriptional regulators are critical for formation and maintenance of follicles. These transcriptional regulators include: Figla, Lhx8, Nobox, Sohlh1, and Sohlh2. A subset of these transcriptional regulators is mutated in women with ovarian insufficiency and infertility. Establishment of this oocyte pool is essential for fertility. This review focuses on these transcriptional regulators of female primordial follicles.