• Molecular & Cellular Toxicology
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• v.1 no.3
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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.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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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.

• Journal of Oriental Neuropsychiatry
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• v.18 no.1
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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.

• Journal of the Korean Society for Marine Environment & Energy
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• v.10 no.2
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• pp.86-92
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• 2007

In this study, we tried to quantitatively study the distribution of estrogenic activity in sediment from Gwangyang Bay by E-screen assay. Besides, we compared the estrogenic activity and the concentration of chemical pollutants. The highest estrogenic activity was recorded at the stations(GY6 and GY8) close to industrial complex and the river mouth of Seomjin. These results obtained from the E-screen assay similar to those of simultaneous analytical detection of 310 chemicals. In particular, GY6 and GY8 sites are confirmed as the full agonist sites because of their RPE values were over 90% having strong estrogenic effect. Also, their EEQ(Estradiol Equivalency Quantity) values are 35.6 ng/g and 14.6 ng/g, low than that of other sites, and these results suggests that have relatively high estrogenic efficiency in Gwangyang Bay. From these results, we can estimate that the stations close to industrial complex and the river mouth of Seomjin are major sources of endocrine disrupter in Gwangyang Bay. On the other hand, when we tried to compare the endocrine disrupter activity and $COD_{Mn}$ value, that is not correlated.

• Environmental Analysis Health and Toxicology
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• v.28
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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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• v.6 no.1
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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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• v.29 no.9
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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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• v.30 no.1
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• pp.7-11
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• 2006

This study was conducted to examine the effects of hormone and sodium pyruvate on in vitro maturation of canine oocytes. Canine oocytes were collected from the ovaries of dogs and cultured in NCSU-37 medium with hormones and sodium pyruvate for 72 hr. Oocytes matured to the metaphase II (MII) stage were observed only from estradiol $17{\beta}\;(E_2)$, and the presence of gonadotropin did not improve the nuclear maturation. No oocytes were developed to the MII stage when $E_2$ was added to medium during the first 6 and 24 hrs of culture period. The presence of $E_2$ during the whole culture period enhanced the nuclear maturation to the MII stage (6.0%, P<0.05). High concentration of sodium pyruvate (2.5 mM) slightly enhanced the nuclear maturation to the metapahse I (HMI) stage, but not the MII stage. the result of the present study shows that the presence of $ E_2$ during the whole culture period of 72 hr enhances the maturation of canine oocytes to the M stage, but sodium pyruvate does not affect the nuclear maturation of the canine oocytes.

• Korean Journal of Acupuncture
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• v.20 no.3
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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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• v.10 no.4
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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.