• Clinical and Experimental Reproductive Medicine
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• 제19권2호
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• pp.105-116
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• 1992

The present study was examined to clarify the role of calcium ion as a factor for the maturation of mouse oocytes. Follicles and cumulus-enclosed oocytes were isolated with two sharp needles under a stereomicroscope from female mouse (ICR) ovaries which were treated PMSG 5 IU 45-46 hours previously. Isolated follicles and cumulus-enclosed oocytes were cultured for 14-16 hours in an organ culture system at $37^{\circ}C$, 5% $CO_2$ in air and 100% humudified in incubator. MHBS was the basic medium used from which A23187, verapamil, $NiCl_{2.}$ $6H_2O$ and $LaCl_{3.}$ $7H_2O$ were added depending on the experimental groups. In follicle- or cumulus-enclosed oocytes wre cultured in these differently treated media. Following results were obtained from the present study. 1. The calcium ionophore A23187 directly or indirectly seems to stimulate GVBD of follicle-enclosed mouse oocytes. Increasing concentration of ionophore A23187 1ed to an increase in oocytes degeneration from the cumulus-enclosed mouse oocytes. 2. The organic $Ca^{++}$ channel blocker, verapamil does not induce GVBD of follicle-enclosed mouse oocytes. Specially, higher dose of 1 mM verapamil induced GVBD of follicle-enclosed mouse oocytes. However, cytoplasm of GVBD oocytes in 1 mM verapamil treated groups appeared shrunk. In the cumulus-enclosed oocytes, polar body formation was reduced in verapamil treated groups and degeneration increased. Verapamil inhibit oocyte maturation (polar body formation). 3. The $Ca^{++}$ inhibitor, Nickel ($NiCl_{2.}$ $6H_2O$) inhibits maturation of the follicle-enclosed oocytes. In the cumulus-enclosed oocytes the progression to MII (PB formation) was reduced and degeneration of mouse oocytes increased as the concentration of $Ni^{++}$ increase. The results indicates that nickel act as an inhibitor of calcium. 4. The $Ca^{++}$ inhibitors, Lanthanum ($LaCl_{3.}$ $7H_2O$) has shown different effect from that of nickel. In follicle-enclosed oocytes, 0.01mM lanthanum induced maturation of mouse oocytes. Polar body formation was reduced in the cumulus-enclosed oocytes all lanthanum treated group.

• 한국발생생물학회지:발생과생식
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• 제12권2호
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• pp.125-132
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• 2008

멜라토닌(N-acetyl-5-methoxytryptamine)은 포유동물의 뇌의 송과선에서 분비되는 호르몬으로 수면과 생체 리듬 등을 조절하고 난소 기능과 번식에도 영향을 미친다. 또한, 강력한 scavenger로서 항산화제의 역할을 한다. 이 연구의 목적은 멜라토닌이 생쥐 난구세포-핵낭(germinal vesicle, GV) 시기 난자 복합체의 체외성숙에 미치는 영향을 알아보는 것이다. 3주령의 ICR 암컷 생쥐의 난소에서 회수된 난자-난구세포 복합체를 0, 0.1 nM, 10 nM, 1,000 nM의 멜라토닌이 첨가된 배양액에서 18시간 동안 배양하고, 제1극체의 방출 여부를 확인하여 성숙율을 확인하였다. 체외성숙 후 TUNEL assay와 성숙율이 가장 높게 나타났다. 체외성숙된 난자에서는 세포자연사가 나타나지 않았으나 난구세포에서는 관찰되었으며, 1,000 nM을 첨가하여 배양한 군의 난구세포는 유의하게 낮은 세포자연사를 나타냈다. 그리고 1,000 nM의 멜라토닌을 첨가한 군의 난구세포에서 멜라토닌 수용체의 mRNA가 대조군에 비해 낮게 발현되었다. 이상의 결과를 종합하면 체외성숙 배양액에 첨가된 멜라토닌은 난구세포의 세포자연사를 줄여줌으로써 생쥐 미성숙 난자의 체외성숙을 향상시키는 역할을 하는 것으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제14권6호
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• pp.759-764
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• 2001

This study was carried out to investigate the effect of activation timing, cell cycle and passage on the development of embryos produced by cumulus cell nuclear transfer in Hanwoo (Korean cattle). Nuclear donor cumulus cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum at $38.5^{\circ}C$ in a humidified atmosphere of 5% $CO_2$ in air. The 1~6 passages of serum deprived or actively dividing cumulus cells were isolated and used as donor cells. The in vitro matured oocytes were enucleated and then the isolated donor cells were introduced. One pulse of 180 volts for $15{\mu}s$ was applied to induce the fusion between karyoplast and cytoplast. The activation was done before or after the fusion. To activate, oocytes were treated with $10{\mu}M$ calcium ionophore for 5 min immediately followed by 2 mM 6-dimethylaminopurine for 3 h. The nuclear transfer embryos were cultured in $500{\mu}l$ of modified CRlaa supplemented with 3 mg/ml BSA in four well dish covered with mineral oil. After 3 days culture, culture medium was changed into modified CRlaa medium containing 1.5 mg/ml BSA and 5% FBS for 4 days. The incubation environment was 5% $CO_2$, 5% $O_2$, 90% $N_2$ at $38.5^{\circ}C$. There was no blastocyst formation when the nuclear transfer embryos were activated before the fusion, whereas, 29.9% of blastocyst formation was shown when the nuclear transfer embryos were activated after the fusion. When serum deprived and actively dividing cumulus cells were used as nuclear donor cells, the developmental rates to blastocyst were 38.5% and 40.6%, respectively. There was no significant difference between serum deprived and actively dividing cells in the developmental rates. The developmental rates to blastocyst according to 1~6 passages were 37.5~44.4%. However, there were no significant differences among passages. These results indicate that 1~6 passage cumulus cell irrespective of cell cycle could support development of nuclear transfer embryos activated after the fusion.

• 한국가축번식학회지
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• 제17권2호
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• pp.141-148
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• 1993

This studies were carried out to investigate the effects of co-culture with cumulus cells, oviduct epithelial cells and uterine endometrium cells on the in-vitro fertilization and cleavage rate of bovine follicular oocytes. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluids from the visible follicles of diameter 3~5 mm. The follicular oocytes were cultured in TCM-199 medium containing hormones and 10% FCS for 24~48 hrs in a incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 12~18 hrs with motile capacitated sperm by preincubation. The results obtained in these experiments were summarized as follows: 1. The in vitro maturatin and fertilization rate of bovine oocytes co-cultured with cumulus cells in TCM-199 medium were 64.0~74.1% and 40.0~58.6% respectively. And in-vitro fertilization rate of cumulus-enclosed oocytes(55.4%) were significantly(p<0.05) higher than cumulus-denuded oocytes(23.1%). 2. The in-vitro maturatin and fertilization rate of bovine oocytes co-cultured with 1$\times$104 cells/ml, 1$\times$106 cells/ml, 1$\times$108 cells/ml and 1$\times$1015 cells/ml oviduct epithelial cells in TCM-199 medium were 59.3% and 40.7%, 64.0% and 48.0%, 58.3% and 37.5%, 52.0% and 32.0%, respectively. 3. The in-vitro maturation and fertilization rate of bovine oocytes co-cultured with 1$\times$104 cells/ml, 1$\times$106 cells/ml, 1$\times$108 cells/ml and 1$\times$1015 cells/ml uterine endometrium cells in TCM-199 medium were 56.0% and 36.0%, 60.7% and 42.9%, 59.3% and 37.0%, 52.0% and 36.0%, respectively. 4. When the in-vitro fertilized oocytes were co-cultured with cumulus cells, oviduct epithelial cells and uterine endometrium cells, the development rate to be blastocyst was 12.2%, 15.6% and 11.7%, respectively and rates were higher than that of control, 2.1%(P<0.05).

• Clinical and Experimental Reproductive Medicine
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• 제25권2호
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• pp.207-216
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• 1998

Certain types of somatic cells, as well as follicular cumulus cells associating with mammalian oocytes, are known to produce beneficial effects on in vitro fertilization and pre implantation development of mammalian eggs when they are present in oocyte culture medium. To investigate the nature of the effects of somatic cells, the resistance of mouse oocytes against chymotrypsin treatment was examined after culture within various cell conditioned media. When mouse oocytes matured for 17-18 hr in the presence of cumulus cells were treated with 1 % chymotrypsin, half of them remained still alive even after 240 min $(t_{50}>240.0)$. In contrast half of mouse oocytes cultured without cumulus cells underwent degeneration within 65.0 min $(t_{50}=65.0{\pm}13.2min)$ of the same treatment. To see if the effects were duc to the secretory products of cumulus cells, mouse cumulus cells were cultured for 20 hr in medium containing 0.4% BSA and the supernatant of culture medium (conditioned medium) was taken. After maturation in the cumulus cell conditioned medium, mouse oocytes exhibited $t_{50}=190.0{\pm}10.8$ min upon chymotrypsin treatment whereas half of oocytes cultured without conditioned medium degenerated within 25.5 min. Human granulosa cell conditioned medium gave similar effects such that oocytes matured in conditioned medium exhibited $t_{50}=183.3{\pm}19.1$ min while $t_50$ of control group oocytes was $60.0{\pm}6.8$ min, Oocytes matured in vero cell conditioned medium exhibited $t_{50}=196.7{\pm}8.8$ min. On the other hand, amniotic cell conditioned medium resulted in the chymotrypsin resistance of $t_{50}=80.0{\pm}8.4$ min which was not statistically different from the control value of $t_{50}=48.0{\pm}13.2$ min. Based upon these results, it is suggested that certain somatic cell types including cumulus cells might change the biochemical properties of mouse oocyte membrane during meiotic maturation as revealed by the enhanced resistance against chymotrypsin treatment. Such effects of somatic cells appear to be mediated via the secretory products rather than direct communication between somatic cells and oocytes.

• Clinical and Experimental Reproductive Medicine
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• 제43권2호
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• pp.82-89
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• 2016

Objective: The long interspersed elements (LINE-1, L1s) are a group of genetic elements found in large numbers in the human genome that can translate into phenotype by controlling genes. Growing evidence supports the role of epigenetic in polycystic ovary syndrome (PCOS). The purpose of this study is to evaluate the DNA methylation levels in LINE-1 in a tissue-specific manner using cumulus cells from patients with PCOS compared with normal controls. Methods: The study included 19 patients with PCOS and 22 control patients who were undergoing controlled ovarian hyperstimulation. After oocyte retrieval, cumulus cells were extracted. LINE-1 DNA methylation levels were analysed by bisulfite treatment, polymerase chain reaction, and restriction enzyme digestion. The Connection Up- and Down-Regulation Expression Analysis of Microarrays software package was used to compare the gene regulatory functions of intragenic LINE-1. Results: The results showed higher LINE-1 DNA methylation levels in the cumulus cells of mature oocytes in PCOS patients, 79.14 (${\pm}2.66$) vs. 75.40 (${\pm}4.92$); p=0.004, but no difference in the methylation of cumulus cells in immature oocytes between PCOS and control patients, 70.33 (${\pm}4.79$) vs. 67.79 (${\pm}5.17$); p=0.155. However, LINE-1 DNA methylation levels were found to be higher in the cumulus cells of mature oocytes than in those of immature oocytes in both PCOS and control patients. Conclusion: These findings suggest that the epigenetic modification of LINE-1 DNA may play a role in regulating multiple gene expression that affects the pathophysiology and development of mature oocytes in PCOS.

• Clinical and Experimental Reproductive Medicine
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• 제25권1호
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• pp.35-41
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• 1998

This study was carried out to investigate the development rates of human embryos co-cultured with cumulus cells to each blastocyst stage. Human zygotes were co-cultured on cumulus cell monolayer in YS medium supplemented with 20% hFF. On day 2, if patient had four or more "good" embryos (regular blastomeres without fragmentation), embryos were further cultured for 72hrs. Blastocysts on day 5 were classified into early blastocyst (ErB), early expanding blastocyst (EEB), middle expanding Blastocyst (MEB), and expanded blastocyst (EdB) on the basis of their morphological aspects of trophectoderm cells and blastocoele. Subsequently, maximum 3 of best blastocysts were transferred in 486 cycles. The results in this study were as follows: Patients who had four or more "good" embryos on day 2 were 498 persons, but patients whose embryos could not be transferred due to failure in development to the blastocyst stage on day 5 were 12 persons (2.4%). The development rate of embryos to the blastocyst stage was 58.2% (2,885/4,957) on day 5, and the rates that developed to the ErB, EEB, MEB, and EdB stage were 15.0% (743/4,957), 14.9% (739/4,957), 14.4% (714/ 4,957), and 13.9% (689/4,957), respectively. Total 1366 blastocysts were transferred in 486 cycles (mean number=2.81). The implantation rate and the ongoing implantation rate obtained by observing the number of G-sac and FHB were 29.9% (409/1,366) and 22.5% (308/1,366), respectively. The clinical pregnancy rate was 51.2% (249/486), and the ongoing pregnancy rate' was 39.1% (190/486). Among women showing ongoing pregnancy, women with singleton were 50% (95/190), women with twin were 37.9% (72/190), and women with triplet were 12.1% (23/190). Although triplet pregnancy rate in this study was high such as 12.1%, because many blastocysts with high viability were produced in our co-culture system using cumulus cells on day 5, we really believe that a multiple pregnancy except twin should not occur by selecting good embryos for maximum two blastocyst transfer. These results demonstrate that autologous cumulus cells may be used for the production of blastocysts with high developmental competence, and the use of autologous cumulus cells to be collected easily, and to be treated conveniently at OPU must be an effective means for obtaining high implantation and pregnancy rate.

• 한국가축번식학회지
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• 제19권2호
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• pp.135-140
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• 1995

The study was conducted to investigate on the survival rate of bisected porcine embryos co-cultured in 10% FCS(v/v)＋TCM-199 media containing hormones, oviductal epithelial cells and cumulus cells 0 to 72 hrs after bisection. In vitro survival rate was defined as development rate on in vitro culture or FDA-test. The results are summarized as follows ; 1. The survival rate of bisected porcine embryos cultrued in 10% FCS＋TCM-199 media contaning PMSG, hCG, PMSG＋hCG, hCG＋$\beta$-estradiol 0 to 20 hrs and 20 to 40 hrs were 37.6% and 37.5%, 28.6% and 28.6%, 35.7% and 28.8%, 30.8% and 23.1%, 38.5% and 30.7%, respectively. The survival rate of bisected embryos co-cultured in TCM-199 media containing hormones, oviductal epithelial cells and cumulus cells was significantly higher than that of non co-culture. 2. The survival rate of bisected porcine embryos co-cultured 10% FCS＋TCM-199 media containing oviductal epithelial cells 4 to 5 hrs and 20 to 24 hrs were 42.9% and 38.5%, respectively. 3. The survival rate of bisected porcine embryos co-cultured in 10% FCS＋TCM-199 media containing cumulus cells 4 to 5 hrs and 20 to 24 hrs were 40.0% and 35.7%, respectively.

• 한국가축번식학회지
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• 제12권2호
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• pp.120-124
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• 1988

Rat oocyte-cumulus complexes were cultured in verious media in order to induce maturation division in vitro. When the complexes were cultured in mKRB containing estrous rat serum (ERS) and FCS of 5% instead of BSA higher proportions(83.3 and 86.7%) of oocytes matured to metaphase II in 20h compared to control(75%) and t도 maturation rates in mKRB plus FCS were generally higher than those in mKRB plus ERS. Fertilization and early cleavage rates in vitro of the intact oocytes matured in mKRB containing BSA and FCS were generally higher than those of cumulus-removed oocytes and these rates were higher in mKRB containing 5% FCS than those in mKRB containing BSA. These results indicate that maturation rate in vitro was greatly increased by the addition of FCS instead of BSA to mKRB solution and the presence of cumulus cells around oocytes prior to sperm insemintion may be responsible for the increase of in vitro fertilization and early cleavage rates.

• 한국환경과학회지
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• 제1권2호
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• pp.15-23
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• 1992

Development of cumulus is studied by numerically integrating the equation of motion equations of conservation for water vapor mixing ratio, and the thermodynamic energy equuation. We use the terrain-following coordinate system called z'-coordinate system, in which we can easily treat any calculation domain with terrain configuration such as mountains. The model domain of calculation is restricted vertically to 4.Skin and horizontally to 100 km, has a bell-type mountain in the centeral part. Four cases are considered, one in a neutral environment, second in a slightly stable environment, third in a environment decreasing water content with low value of initial water vapor mixing ratio, the fourth in a case with higher vapor gradient. The more the atmosphere is unstable, the more cumulus develops easily and the more water vapors is abundant, the more cumulus develops easily too. More detailed cloud microphysics parameterizations and wet deposition must be conridered to use in air pollutants prediction model.