• Biomolecules & Therapeutics
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• 제13권1호
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• pp.7-12
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• 2005

GyeongshinhaeGihwan T1 (GGT1) is a newly developed oriental medicine to help weight control. We investigated nitric oxide production and cytokine secretion in mouse peritoneal macrophages. According to recent reports, macrophages are participated in fat accumulation and closely related with obesity. In this study, using mouse peritoneal macrophages, we have examined whether GGT1 affects the production of nitric oxide (NO), tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$), and interleukin (IL)-12 by the stimulation of interferon-${\gamma}$ and lipopolysaccharide (LPS). GGT1 inhibits LPS-induced NO production in a dose-dependent manner. The decrease in NO synthesis was reflected as a decreased amount of inducible NO synthese protein. We also found that GGT1 inhibits pro-inflammatory cytokines, TNF-${\alpha}$ and IL-12 production. In mouse embryo preadipocyte 3T3-L1, GGT1 reduced the viability in a dose-dependent manner. These findings suggest that GGT1 may have potential effects in preventing and controlling adipogenesis and obesity.

• 한국발생생물학회지:발생과생식
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• 제16권3호
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• pp.219-226
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• 2012

It is suggested that carbohydrate metabolites may involve in the development of morula to blastocyst but many of the mechanisms are not unmasked. Two-cell stage embryos were collected and examined the effects of lactate on the development of blastocyst in vitro. The expression profiles of lactate dehydrognase (Ldh) genes and aquaporin (Aqp) genes were analyzed with RT-PCR. The successful development from morula to blastocyst was dependent on lactate concentrations. The expression profiles of Ldh genes were changed by the lactate concentration. Ldha was expressed in morula stage at 10 mM lactate, and in blastocyst stage at lactate free condition. Ldhb was expressed in morula stage at 10 mM and 20 mM lactate, and in blastocyst stage at 10 mM lactate. Aqp genes were also showed different expression patterns by the lactate concentrations. Aqp3 was expressed in hatching embryo at 120 hr post hCG administration (hph) which was cultured in BWW medium and lactate free condition. Aqp7 was expressed in hatching embryos at 120 hph which was cultured at 10 mM lactate condition. Also Aqp8 was expressed in hatching embryo at BWW and 20 mM lactate condition. Aqp9 was expressed in morula at BWW and 10 mM lactate condition, and in blastocyst at BWW. Based on these results, it is suggested that concentration of lactate in the medium and the level of lactate synthesis in embryo is critical factor for blastocoels formation. In addition it is suggested that LDH may involve the AQPs expression in embryos.

• 한국수정란이식학회지
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• 제26권3호
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• pp.201-207
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• 2011

The present study was performed to investigate the survival and subsequent embryonic developmental rate of immature and mature oocytes after vitrification and pronuclear stage embryos after slow-freezing and vitrification. We have also tried to examine the dependency of concentrations (7.5, 15%) and exposure time (5, 10, 20 min) of ED cryoprotectant on developmental rate of pronuclear stage embryos. The developmental rates of 2-ce1l and blastocyst embryos at mature oocytes were significantly (p<0.05) higher than immature oocytes. After slow freezing, vitrification and thawing of pronuclear stage embryo, the survival and developmental rates of blastocysts and hatched blastocysts were significantly (p<0.05) higher after vitrification than after slow-freezing. On contrary, the developmental rates of 2-cell embryos were significantly (p<0.05) higher after slow freezing than after vitrification. The cryopreservation methods of pronuclear stage embryos vitrified by exposed to 7.5% ED solution for 5 minutes was significantly (p<0.05) higher than other experimental group. The results of our study suggest 1hat the developmental rates of mature oocytes have been more successful than immature oocytes during vitrification. Vitrification was more efficient than slow freezing in case of pronuclear stage embryos. The effective cryopreservation method of pronuclear stage embryos was vitrified by exposed to 7.5% ED solution for 5 minutes.

• 한국수정란이식학회지
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• 제12권3호
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• pp.283-291
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• 1997

In vitro development of bovine embryos is affected by many factors such as energy substrates, amino acids, and some growth factors. It has been reported that mRNA of insulin, PDGF and their receptors are detected in cow embryos, and that some chelating agents such as EDTA and transferrin have beneficial role on mouse and bovine embryos. The author hypothesized that insulin, transferrin arid PDGF added to a culture medium increase in vitro development of bovine embryos by chelating toxic substance(s) or increasing cell growth and metabolism. Immature oocytes from slaughtered ovaries of Holstein cows and heifers were matured for 24 hours in a TCM199 containing 10% fetal calf serum, FSH, LH and estradiol with granulosa cells in vitro. Matured oocytes were coincubated with sperm for 30 hours in a modified Tyrode's medium (IVF). Embryos cleaved to 2- to 4-cell at 30 hours after IVF were selected and cultured in a 30-$\mu$l drop of a synthetic oviduct fluid medium (SOFM) containing 0.8% BSA, Minimum Essential Medium essential and non-essential amino acids, and insulin, transferrin or PDGF for 9 days. Supplementation of a SOFM with insulin, and /or transferrin did not increase develop-mental rate to expanding and hatching blastocyst of 2- to 4-cell bovine embryos compared with control. The highest developmental rate to hatching blastocyst was shown when PDGF was added at the concentration of 10 ng /ml among the supplementing doses tested in the present study (p<0.05). Addition of PDGF without insulin to a SOFM could not increase embrye development, but combined addition of PDGF with insulin significantly increased (p<0.05) embryo development to hatching blastocyst (50%) compared with control (38%). In conclusion, insulin and PDGF supplemented to a SOFM may act synergistically and have beneficial effect on in vitro development of 2- to 4-cell bovine embryos matured and fertilized in vitro.

• Reproductive and Developmental Biology
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• 제36권4호
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• pp.255-260
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• 2012

The purpose of this study was to assess follicular viability and competence through in vitro culture of preantral follicles isolated from vitrified mouse whole ovaries. Mouse preantral follicles were enzymatically isolated from vitrified- warmed and fresh ovaries and cultured for 10 days followed by in vitro oocyte maturation. In vitro matured oocytes were fertilized and cultured to the blastocyst stage. Five minutes pre-exposure to vitrification solution of whole ovaries had significantly higher (p<0.05) oocyte survival and maturation rates than between 10 min exposure groups. Oocyte diameter was significantly smaller (p<0.05) in the 5 and 10 min exposure groups ($69.4{\pm}2.8$ and $67.8{\pm}3.1$) when compared to that of control group ($71.7{\pm}2.1$). There was no statistical significant difference in blastocyst development rates between vitrification group (8.6%) and the fresh control group (12.0%). The mean number of cells per blastocyst was significantly lower (p<0.05) in the vitrification group ($41.9{\pm}20.2$) than in the fresh control group ($55.1{\pm}22.5$). The results show that mouse oocytes within preantral follicles isolated from the vitrified whole ovaries can achieve full maturation, normal fertilization and embryo development.

• Reproductive and Developmental Biology
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• 제29권2호
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• pp.117-120
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• 2005

This study was conducted to establish the optimal temperature condition before oocyte activation in B6m F1 mouse. In experiment 1, two embryo culture media (CZB vs KSOM) were evaluated for the development of activated mouse oocytes. Parthenogenetic embryos cultured in KSOM showed better blastocyst development than ones cultured in CZB $(56.2\%\;vs\;81.0\%\;p<0.01)$. Two-hour of pre-incubation before activation significantly reduced the number of hatched blastocysts in KSOM $(22.0\%\;versus\;8.8\%\;p<0.05)$. In experiment 2, recovered oocytes were pre-incubated at different temperature conditions before activation. The experimental groups were divided by 5 as follows. Group A: pre-incubation for 120 min at $37^{\circ}C$, Group B: pre-incubation at $37^{\circ}C$ for 90 min then at $25^{\circ}C$ for 30 min, Group C: pre-incubation at $37^{\circ}C$ for 60 min then at $25^{\circ}C$for 60 min, Group D: pre-incubation at $37^{\circ}C$ for 30 min then at $25^{\circ}C$ for 90 min, and Group E: pre-incubation at $25^{\circ}C$ for 120 min before activation. Group A $(67.6\%)$ and B $(66.7\%)$ showed better development to the blastocyst stage than other groups $(Group\;C:\;50.0\%\;Group \;D:\;49.2\%\;Group\;E:\;33.3\%,\;p<0.05)$. The present study indicates that the temperature before activation affects the development of B6D2 F1 mouse parthenogenetic oocytes and exposure to room temperature should be limited to 30-min when the oocytes are left in HEPES-buffered medium for micromanipulation.

• 한국임상수의학회지
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• 제29권1호
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• pp.27-32
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• 2012

The purpose of this study was to evaluate survival rates of vitrified mouse blastocysts in various vitrification solutions (cryoprotectants) and apparatuses. The mouse blastocysts were harvested from culture of mouse 2 cell embryo and were divided into three group (i) untreated (control); (ii) exposed to cryoprotectant agents; or (iii) cryopreserved by various vitrification apparatuses. Vitrification solutions are 40% ethylene glycol (EG) + 5.8 mg/mL ficoll + 0.5M sucrose (EFS solution), 3M glycerol + 3M EG (ES solution), 20% EG + 20% dimethyl sulfoxide (ED solution), 3M EG + 1.0 m sucrose (ES solution). Vitrification apparatuses consisted of 5 groups ; closed plastic straw (CPS), electron microscope (EM) grid, cryoloop, open pulled straw (OPS), and glass micropippete in plastic straw (GPS). The survival rates of control were 88.0%. The survival rates of exposed blastocysts in EFS, GE, ED, and ES solutions were 70.8%, 43.5% (P<0.01), 83.3% and 65.2%, respectively. The survival rates of vitrified blastocysts in CPS, EM grid, cryoloop, OPS and GPS were 56.5% (P< 0.01), 72.7%, 83.3%, 60.9% (P<0.05) and 54.2% (P<0.01), respectively. Among the vitrification solutions, the highest survival rate was seen in blastocysts vitrified in EG + DMSO (83.3%). The survival rate was not significantly different from that of the control (88%). Blastocysts cryopreserved with glycerol in all groups had an overall low survival rate of 43.5%. Survival rate of mouse blastocysts between vitrification apparatuses showed higher in cryoloop.

• 한국가축번식학회지
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• 제18권3호
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• pp.199-206
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• 1994

본 연구는 생쥐 배 발생과정의 상이한 발현을 RT-PCR법에 의해 무작위 증폭함으로서 새로운 유전자를 손쉽게 크로닝하기 위해 수행되었다. mRNA의 상이한 display법은 Ling 과 Pardee (Science 257, 1992)에 의해 개발되었으며, 최근 Zimermann과 Schultz (PNAS USA 91, 1994)에 의해 재증명되었다. 이 방법은 특정 유전자의 일시적 발현의 변화가 maternal 제어로부터 접합체 제어로의 이행에 따른 발현전이, 다정자 침입과 단일 정자 침입에 의한 배발생의 기능적 차이, 성공적으로 부화한 배반포기 배와 부화에 실패한 배반포기 배에서의 발현의 차이는 물론 세포주기에 따른 유전자 발현 양식의 변화에 따른 새로운 유전자의 크로닝을 가능케 한다. 이 방법에 의해, 2세포기 특이 발현 유전자를 크로닝 하였으며, 이 유전자는 EcoRI제한 효소 처리후 Southern blot을 행한 결과 약 15kb genomic size를 가진 것으로 나타났다. 이 새로운 유전자는 간장 특이적 발현을 나타내었다. 또한, 적어도 2개의 mRNA가 존재하였으며, 이는 RNA splicing에 의한 것으로 추정되었다. (PCR, RT-PCR, cloning, preimplantation, mouse)

• 한국가축번식학회지
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• 제16권3호
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• pp.269-276
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• 1992

Studies were carried out to find the freezing media which gives no ice crystals in single(glycerol, ethylene glycol, dimethyl sulfoxide(DMSO)) and mixture solutions(glycerol＋propylene glycol, glycerol＋ethylene glycol) of permeable cryoprotectants in vitrification solution and to study effects of VS on the survival of vitrified mouse morulae. The results are summarized as follows: 1. In toxicity test of permeable cryoprotectants, 30% glycerol of single solution showed the highest FDA-score(4.1) in mouse morulae frozen compared among other single solutions. The FDA-score(4.1) of 30% glycerol was higher than 30% ethylene glycol(3.6) and DMSO(1.4( (P<0.05). 2. 20, 30 or 40% single solution of permeable cryoprotectants containing m-PBS with 10% sucrose and 20% BSA was not crystallized during cooling, but crystallized during warming. However, the 30% mixture solution of the two permeable cryoprotectants was not crystallized both during cooling and warming.3. When mouse morulae were frozen in 30% mixture solutions of two permeable cryoprotectants(glycerol and propylene glycol, glycerol and ethylene glycol), highest FDA-score(4.5) was obtained in a mixture solution of 20% glycerol and 10% ethylene glycol(20G10E) than other 30% mixture solutions(10G20E, 15G15E, 20G10P, 15G15P, 10G20P) and there was significant difference between 20G10E and 10G20E(P<0.05).

• Clinical and Experimental Reproductive Medicine
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• 제17권2호
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• pp.167-172
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• 1990

This study was carried out to clarify the effects of various kinds of cryoprotectants which were frequently used in freezing embryos of domestic animals on the survival of frozen-thawed mouse embryos. Mouse embryos were collected by hyperstimulation induction of ICR mouse. The samples were slowly cooled ($l^{\circ}C/min$) to temperatures between $-7^{\circ}C$ and $-30^{circ}C$ before direct transfer to liquid nitrogen ($-196^{\circ}C$) and thawed rapidly ($-500^{\circ}C$/min). As cryoprotectants, Glycerol, DMSO, Ethylene glycol and Propylene glycol were used and applied each 2 cell, 8 cell, morula in embryo stage. After normal mouse embryos developed to blastocyst by in vitro culture, we observed recovery rate and developing rate of embryos at thawing. The results obtained in these experiments were as follows : 1. The in vitro development rate from the frozen-thawed 2 cell embryos to the blastocyst were 67.7% in ethylene glycol, 65.7% in Propylene glycol, 55.2% in glycerol and 50.0% in DMSO respectively. 2. The in vitro development rate from the frozen-thawed 8 cell embryos to the blastocyst were 83.6% in DMSO, 75.7% in glycerol, 52.2% in propylene glycol respectively. 3. The in vitro development rate from the frozen-thawed morula to the blastocyst were 84.2% in glycerol, 80.0% in DMSO, 66.6% in propylene glycol and 55.2% in ethylene glycol respectively.