• Molecular & Cellular Toxicology
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• 제1권3호
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• pp.149-156
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• 2005

Di (n-ethylhexyl) phthalate (DEHP) is thought to mimic estrogens in their action, and are called endocrine disrupting chemicals. DEHP is used in numerous consumer products, especially those made of flexible polyvinyl chloride and have been reported to be weakly estrogenic. In this study, DEHP were tested for estrogenic properties in vitro models and with microarray analysis. First, the E-screen assay was used to measure the proliferation of DEHP in MCF-7 cells, a human breast cancer cell line. DEHP induced an increase in MCF-7 cell proliferation at concentration of $10^{-4}M$. Second, we carried out a microarray analysis of MCF-7 cells treated with DEHP using human c-DNA microarray including 401 endocrine system related genes. Of the genes analyzed, 60 genes were identified showing significant changes in gene expression resulting from DEHP. Especially, 4 genes were repressed and 4 genes were induced by DEHP compared to $17{\beta}-estradiol$. Among these genes, trefoil factor 3 (intestinal), breast cancer 1, early onset and CYP1B1 are involved in estrogen metabolism and regulation. Therefore it suggests that these genes may be associated with estrogenic effect of the DEHP on transcriptional level. The rationale is that, as gene expression is a sensitive endpoint, alterations of these genes may act as useful biomarkers to define more precisely the nature and level of exposure to kinds of phthalates.

• 한국가축번식학회지
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• 제21권2호
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• pp.103-109
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• 1997

The studies on the carried out to investigate to determine the optimum thawing temperature and equilibration time and 1 step straw method of frozen bovine embryos. The follicular oocytes were cultured in TCM-199 medium containing 10 IU/ml PMSG(Sigma, USA), 10 IU/ml hCG(Sigma, USA), 1 $\mu\textrm{g}$/ml $\beta$-estradiol(Sigma, USA) and 10% FCS for 24~48 hrs in 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 of heparin. The bovine embryos following dehydration by cryoprotective agents and various concentration of sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water. Survival and in vitro developmental rate was defined as developmental rate on in vitro culture or FDA-test. The results are summarized as followes : 1. The equilibration time on in vitro developmental rates of bovine embryos was attained after short period of time(2.5~5 min.) in the freezing medium higher than long period of time (10~20 min.). 2. The temperature thawed at 3$0^{\circ}C$ after rapid freezing of bovine embryos resulted in a significantly higher in vitro developmental rate than did at 2$0^{\circ}C$ and 35$^{\circ}C$. 3. The thawing time on in vitro developmental rates of bovine embryos was attained after short period of time(1~5 min.) in the freezing mediuim higher than long period of time(10min.). 4. The in vitro developmental rates of bovine embryos after rapid frozen-thawing by 1 step straw method in the freezing medium added 1.5M, 2.0M glycerol, DMSO, propanediol and 0.25M, 0.50M, 0.75M, 1.00M sucrose were 12.5~19.4%, 10.0~15.6%, 9.1~13.8% and 6.7~12.9%, respectively.

• 동의신경정신과학회지
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• 제18권1호
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• pp.15-36
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• 2007

Objective : This study was to investigate the effects of several herbs on the levels of MT1 and MT2 melatonin receptors Methods: It was investigated the effects of several herbs such as WEDL, WEZV, WEFO, WEOC on the levels of MT1 and MT2 melatonin receptors using C6 glial cell model. ${\beta}-estradiol$ treatment, as a positive control group, under non-cytotoxic condition. Results : 1. The water extracts of Dimocarpus long (WEDL) induced the levels of MT2 melatonin receptor expression in a concentration-dependent manner without altering the levels of MT1 melatonin receptor expression. 2. The treatment with the water extract of Zizyphus vulgaris (WEZV) induced the levels of MT1 melatonin receptor expression and the levels of MT2 melatonin receptor expression was not affected. 3. The levels of MT1 as well as MT2 melatonin receptor expression were markedly up-regulated in the water extract of Fossilia ossis (WEFO) and the water extract of Ostreae caro (WEOC)-treated C6 cells. 4. The combination treatment with WEDL and WEZV induced not only the levels of MT1 melatonin receptor expression but also MT2 melatonin receptor expression, but the synergic effects of the combination treatment with WEFO and WEOC were not detected in C6 cells. Conclusion : The study provides important new insights into the possible mechanisms on the regulation of melatonin receptor synthesis by WEDL, WEZV, WEFO and WEOC.

• 한국해양환경ㆍ에너지학회지
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• 제10권2호
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• pp.86-92
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• 2007

본 연구는 E-screen assay를 이용해서 광양만 연안해역 퇴적물의 에스트로겐 활성분포를 정량적으로 평가하고 화학분석 결과와 비교 검토하고자 하였다. 가장 높은 에스트로겐 활성을 보인 지점은 산업단지와 섬진강 유입구에 인접한 G6, G8 이었다. E-screen assay로 얻어진 이러한 결과는 다성분 동시분석 결과와도 일치하였다. 특히 GY6, GY8 지점의 RPE는 90% 이상으로 강한 에스트로겐 작용을 나타내어 full agonist 지점으로 확인되었다. 또한 이들 지점은 EEQ가 각각 35.6 ng/g, 14.6 ng/g로 타 지점에 비해 낮아 에스트로겐 활성 효능이 상대적으로 높을 것으로 판단된다. 이러한 결과로부터, 산업단지와 섬진강 유역에 인접한 지점들을 광양만 연안 해역의 내분비계 교란 물질의 유입원으로 추정할 수 있다. 한편, 에스트로겐 활성도와 화학오염물질과의 비교결과는 상관성이 없는 것으로 나타났다.

• Environmental Analysis Health and Toxicology
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• 제28권
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• pp.16.1-16.8
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• 2013

Objectives Fish vitellogenin (VTG) is produced in the female liver during oogenesis through the estradiol cycle and produced in the male liver by endocrine disrupting chemicals (EDCs) such as alkylphenols. In this study, we propose that the VTG concentration in the pale chub could be detected using monoclonal antibodies and polyclonal antibodies against vitellin (Vn) in a VTG enzyme-linked immunosorbent assay (ELISA) system. Methods Monoclonal antibodies and polyclonal antibodies were produced using the Vn extracted from the matured ovum of the ovary. The VTG was extracted from the plasma of the male pale chub. The Vn and VTG were confirmed by measuring the molecular weight of their proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the specificity of the antibodies was checked through western blotting methods. The assay system was validated with respect to optimal assay concentrations, specificity, recovery, and intra- and inter-assay variations. Results The Vn consisted of two protein bands with apparent molecular weights of 64 and 37 kDa. The SDS-PAGE indicated protein weights of 146 and 77 kDa in the VTG. The assay range was 15.6 ng/mL to 2,000 ng/mL, and the value of the intra- and inter-assay variations were within 10.0% and 14.7%, respectively. The recovery rate was $99.5{\pm}5.5%$. Conclusions A sandwich ELISA was developed that could be used to qualify the VTG of pale chub in screening for EDCs. Pale chub is an ideal species for observing estrogen activity in the environment because of its extensive habitat and extensive food chain. The ELISA developed here would be more favorable than those for other species for determining the effect of long-term food chain accumulation of EDCs in aquatic environments.

• Plant Resources
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• 제6권1호
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• pp.27-35
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• 2003

In this study, as preliminary research for the development of natural estrogen supplement the chemical properties of Iranian black pomegranate juice concentrate and seeds were evaluated. Proximate compositions of pomegranate juice concentrate and seeds were as follows; crude lipid 0.4% and 8.2%, moisture 39.9% and 6.6%, crude protein 0.9% and 12.2%, ash 1.4% and 1.7%, and carbohydrate 42.0% and 84.5% respectively. Major amino acids are glutamic acid (1310.0ppm) and aspartic acid (896.2ppm) in juice concentrate, and glycine (611.1ppm) and arginin (401.6ppm) in seeds. Ascorbic acid has the highest concentration of 20.0mg/l00g in juice concentrate and 0.23mg/l00 in seeds. The compositions of unsaturated fatty acids such as linoleic acid and linolenic acid were higher than those of saturated fatty acids such as stearic palmitic acid. Major minerals were potassium, calcium and sodium, potassium was highest in both juice concentrate and seeds. Vitamins were composed of ascorbic acid (20.0mg/l00g), vitamin B$_1$(0.12mg/100g) and niacin (0.80mg/l00g) in juice concentrate, and only ascorbic acid(0.23mg/l00g) in seeds. Organic acids such as citric and L-malic acid were detected only in pomegranate juice concentrate. The contents of total polyphenols were 4.55g/L in juice concentrate and 3.5mg/l00g in seeds, respectively. Phytoestrogens detected in pomegranate juice concentrate and seeds were daidzein, quercetin, genistein and 17 $\beta$-estradiol.

• Asian-Australasian Journal of Animal Sciences
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• 제29권9호
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• pp.1309-1313
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• 2016

Metabolic signals and the state of energy reserves have been shown to play a crucial role in the regulation of reproductive function. This study was carried out to investigate the effects of dietary glucose levels on puberty onset in gilts. Weight-matched, landrace gilts (n = 36) $162{\pm}3days$ old, weighing about $71.05{\pm}4.53kg$, were randomly assigned to 3 dietary treatment groups of 12 gilts each. The trial lasted until the onset of puberty. Gilts in each group were supplied with diets containing different levels of glucose as follows: i) starch group (SG) was free of glucose, contained 64% corn derived starch; ii) low-dose group (LDG) contained 19.2% glucose and 44.8% corn derived starch; iii) high-dose group (HDG) contained 30% glucose and 30% corn derived starch. Results indicated: i) The growth performance of gilts were not affected by the addition of glucose, but the age of puberty onset was advanced significantly (p<0.05); ii) Compared with the SG, the concentration of insulin significantly increased before puberty in HDG (p<0.05); iii) There was no difference in serum progesterone (P) levels amongst the different feed groups, however, levels of estradiol ($E_2$), luteinizing hormone, and follicle-stimulating hormone were significantly higher at puberty onset in HDG (p<0.05). Overall, our findings indicate that glucose supplementation significantly advances puberty onset, which can have practical purposes for commercial breeding.

• Reproductive and Developmental Biology
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• 제30권1호
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• pp.7-11
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• 2006

본 연구는 호르몬과 Na-pyruvate의 첨가가 개 미성숙난자의 체외성숙에 미치는 영향을 검토하였다. 개의 발정주기와 관계없이 암캐의 난소에서 회수한 난자를 NCSU-37 배양액에서 72시간 체외 배양하였다. 호르몬의 영향을 검토하기 위하여 배양액 중에 다양한 호르몬을 $24{\sim}72$시간 첨가하여 성숙율을 검사하였으며, Na-pyruvate 첨가 농도의 영향을 검토하였다. $E_2$ 단독 첨가구에서는 제2감수분열 중기(MII) 단계까지 성숙이 관찰되었으나(5.7%), 타 처리구에서는 MII기까지의 성숙이 이루어지지 않았다(P<0.05), $E_2$를 $6{\sim}24$시간만 처리하였을 때는 MII기로 성숙되는 난자가 없었으나 72시간 처리하였을 때는 6.0%로 유의적으로 높게 나타났다(P<0.05). Na-pyruvate의 농도를 0.25 mM 첨가한 구보다 2.5 mM 첨가한 구에서 제1감수분열 중기(MI) 단계까지의 성숙율이 증가하는 경향을 보였으나, MII 단계까지의 성숙율은 차이가 없었다. 본 실험의 결과는 개 난자의 체외성숙시 $E_2$가 첨가된 배양액에서 72시간 배양시에 성숙율을 증진시킬 수 있으나, Na-pyruvate의 농도는 개 체외배양에 있어 큰 영향을 미치지 않는 것을 나타낸다.

• Korean Journal of Acupuncture
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• 제20권3호
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• pp.1-8
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• 2003

Objectives : To investigate the increasing effect of veterinary acupuncture (VA) on the eggs-laying rate, we carried out the experiment in old hens. Methods : Twenty four old hens (ISA Brown; 1,200 - 1,400 g) over 75 weeks old whose eggs-laying rates were under the 50% for recent 15 days were used. In experimental group, VA treatments were performed seven times during 10 days. Unang point at the end of bilateral wing and Migun point at the tail bone were stimulated about 30 seconds by the stainless steel needle (0.3 mm o.d.). In control group, similar manual stress were performed at same frequency. The numbers of laid eggs were observed for 26 days after the beginning of treatment. Bloods were sampled two times from wing veins during and after the treatment period and serum concentrations of luteinizing hormone (LH) and Estradiol were detected. Results : Eggs-laying rates of experimental group $(34.29{\pm}2.79%)$ were significantly increased than that of control group $(27.56{\pm}2.00%,\;p<0.05)$ with the significant increasing of LH serum concentration in experimental group $(from\;0.77{\pm}0.13\;IU/I\;to\; 1.42{\pm}0.22\;IU/I,\;p<0.05)$. Conclusions : Increasing effect of VA treatment on the eggs-laying rate in old hens were proved in this study, and it may be the first prompt on the experimental investigation and development of VA.

• Animal cells and systems
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• 제10권4호
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• pp.211-217
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• 2006

Heavy metals are well known as important environmental pollutants and also considered as endocrine disrupters. This study was performed to evaluate the direct effects of heavy metals such as cadmium (Cd), zinc (Zn), mercury (Hg), lead (Pb), cobalt (Co), and arsenic (As) on the various steroidogenic enzymes in frog ovarian follicles. Ovarian follicles from Rana catesbeiana were isolated and cultured for 18 hours in the presence of frog pituitary homogenate (FPH, 0.05 gland/ml) or various steroid precursors with or without heavy metals (0.01-100 ${\mu}M$), and steroid levels in the follicle or culture medium were measured by radioimmunoassay (RIA). Thus, the steroidogenic enzyme activities were indirectly evaluated by measuring the converted steroid levels from the added precursor steroid. Among heavy metals, Hg, Cd and Zn significantly inhibited FPH-induced pregnenolone ($P_5$) production by the follicles ($EC_{50},\;4.0{\mu}M,\;25.6{\mu}M\;and\;5.7{\mu}M$, respectively ), and also suppressed the conversion of testosterone (T) to estradiol $17{beta}\;(E_2)\;(EC_{50},\;4.2{\mu}M,\;7.5{\mu}M\;and\;80.0{\mu}M) while Pb, Co and As are not or less effective in the inhibition. Other enzymes such as $C_{17-20}$ lyase and $17{\beta}$-hydroxysteroid dehydrogenase ($17{\beta}$-HSD) were suppressed only in the high concentration of Hg, Cd and Zn. Taken together, these data demonstrate that cytochrome P450 side chain cleavage (P450scc) and aromatase are much more sensitive to heavy metals than other steroidogenic enzymes and Hg, Cd and Zn show stronger toxicity to follicles than other heavy metals examined.