• Environmental Mutagens and Carcinogens
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• v.26 no.2
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• pp.53-58
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• 2006

Normally, environmental toxicants are classified as endocrine disruptors if they interfere with regulation of cellular function by endogeneous steroids through inhibition of receptor binding and/or transcriptional activation. So, many studies have been performed about agonist/antagonist of hormone receptor to study mechanisms of endocrine disruptors. If toxicants affect steroid biosynthesis and/or degradation and alter hormone homeostasis, these also are classified as endocrine disruptors. But there are not many studies of the mechanisms of endocrine disruptors on the basis of alteration of steroid biosynthesis and/or degradation. Isolation and culture of Leydig cells from testis is one of methods for the steroidogenesis screening assays to evaluate a substance for altering steroidogenesis. Leydig cells were harvested using the method described by Klinefelter with modifications. Leydig cells were purified by perfusion of testis and incubation ($34^{\circ}C$, 80cycles/minute, 20 minutes) with collagenase (0.25 mg/kg), centrifugal elutriation, percoll gradient centrifugation and BSA multidensity gradient centrifugation. To confirm if this method is one of appropriate tools to evaluate a substance for altering steroidogenesis, ketoconazole, positive control was administered to purified Leydig cells. Ketoconazole ($10^{-8}M$ and above) significantly reduced testosterone production in purified Leydig cells. From above results, we suggest that this method for steroidogenesis screening assay appears to be a appropriate tool to detect suspected compounds for altering steroidogenesis.

• Journal of Sericultural and Entomological Science
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• v.30 no.1
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• pp.25-32
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• 1988

In order to examine a physiological role of juvenile(JH) binding proteins in the hemolymph of the silkworm larva, Bombyx mori, [3H] JH I incubated hemolymph was separated by polyacrylamide gel electrophoresis in the fifth-instar larva and the activity of the binding protein was analyzed using charcoal binding assay. The results obtained were as follows; 1. The JH was bound by two protein fractions in the hemolymph of the fifth-instar larva; One was JH binding lipoprotein(JH-LP), the other was JH speific binding protein(JHBP). Their relative mobility values(Rm) were 0.3∼0.33 and 0.81∼0.84, respectively. There were no valid differences in those values from developmental stages of both male and female silkworms. 2. Total protein contents of the hemolymph were gradually increased during the fifth-instar larva, while at the prepupa decreased. The maximum ones were observed at the spinning period and the contents from female were much higher than those from the male. 3. JH binding activity per ml of the hemolymph was low in the early stage of the fifth-instar larva and its activity was maximized at the psinning period and at the prepupa slightly decreased. 4. There was a similar pattern between changes of the JH binding activity per ml of the hemolymph and of the total protein contents of the hemolymph. 5. The JH binding activity per mg of the hemolymph proteins was high in the early stage of the fifth-instar larva, while from the 6th day of the fifth-instar larva to the prepupa its activity showed the lowest levels.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.8
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• pp.1193-1198
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• 2003

An increase in frequency of administration of exogenous growth hormone (GH) or GH-releasing hormone was reported to be a model to increase blood circulating insulin-like growth factor-1 (IGF-1) and to improve growth performance in animals. We have investigated the effect of twice daily administration of GH-releasing peptide-2 (GHRP-2) on growth performance, GH responsiveness and plasma insulin-like growth factor IGF-1 in swine. We administered to eight swine, 3 control and 5 treatment, a twice daily s.c. injections of GHRP-2 ($30{\mu}g/kg\;BW$) for a period of 10 days. Every day blood samples immediately taken before injections of GHRP-2 or saline, at 08:00 h and 16:00 h, were measured for IGF-1 concentrations. Blood samples for GH assay were collected every 20 min on days 1, 6 and 10, from 1 hour before and 3 h after GHRP-2 or saline injections at 08:00 h. GH peak concentrations and GH area under curve (GH AUC) on day 1, 6 and 10 in treatment group of swine were higher than those in control swine (p<0.05). Twice daily administration of GHRP-2 caused a significantly attenuation (p<0.05) of GH peak concentrations ($80.25{\pm}13.87$, $39.73{\pm}5.72$ and $27.57{\pm}6.06ng/ml$ for day 1, 6 and 10, respectively) and GH AUCs ($3,536.15{\pm}738.35$, $1,310.31{\pm}203.55$ and $934.37{\pm}208.99ng/ml$ for day 1, 6 and 10, respectively). However, there was no significant difference in GH peak concentration and GH AUC between day 6 and 10. Plasma IGF-1 concentration levels were higher in treatment than control group of swine (p<0.05) after 3 days of the treatment, and the levels reached a plateau from day 3 to 10 of experiment. Growth performance did not alter by GHRP-2 administration, even though a numerical increase of body weight gain and feed efficiency was observed. These results indicate that twice daily administration of GHRP-2 for 10 days in swine did not significantly influence on growth performance, caused an overall attenuation of GH response, and that elevation of plasma GH concentrations caused by GHRP-2 administration increased plasma IGF-1 concentrations, even though an attenuation of GH response was observed.

• Environmental Analysis Health and Toxicology
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• v.31
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• pp.10.1-10.8
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• 2016

Objectives Aromatase inhibitors that block estrogen synthesis are a proven first-line hormonal therapy for postmenopausal breast cancer. Although it is known that standardized extract of Ginkgo biloba (EGb761) induces anti-carcinogenic effects like the aromatase inhibitors, the effects of EGb761 on steroidogenesis have not been studied yet. Therefore, the effects of EGb761 on steroidogenesis and aromatase activity was studied using a H295R cell model, which was a good in vitro model to predict effects on human adrenal steroidogenesis. Methods Cortisol, aldosterone, testosterone, and $17{\beta}$-estradiol were evaluated in the H295R cells by competitive enzyme-linked immunospecific assay after exposure to EGb761. Real-time polymerase chain reaction were performed to evaluate effects on critical genes in steroid hormone production, specifically cytochrome P450 (CYP11/ 17/19/21) and the hydroxysteroid dehydrogenases ($3{\beta}$-HSD2 and $17{\beta}$-HSD1/4). Finally, aromatase activities were measured with a tritiated water-release assay and by western blotting analysis. Results H295R cells exposed to EGb761 (10 and $100{\mu}g/mL$) showed a significant decrease in $17{\beta}$-estradiol and testosterone, but no change in aldosterone or cortisol. Genes (CYP19 and $17{\beta}$-HSD1) related to the estrogen steroidogenesis were significantly decreased by EGb761. EGb761 treatment of H295R cells resulted in a significant decrease of aromatase activity as measured by the direct and indirect assays. The coding sequence/Exon PII of CYP19 gene transcript and protein level of CYP19 were significantly decreased by EGb761. Conclusions These results suggest that EGb761 could regulate steroidogenesis-related genes such as CYP19 and $17{\beta}$-HSD1, and lead to a decrease in $17{\beta}$-estradiol and testosterone. The present study provides good information on potential therapeutic effects of EGb761 on estrogen dependent breast cancer.

• Development and Reproduction
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• v.12 no.2
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• pp.125-132
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• 2008

Melatonin (N-acetyl-5-methoxytryptamine), a major hormone of pineal gland in vertebrates, is known to be associated with regulation of the dynamic physiological functions in general and has some functions on reproduction in the ovarian follicles in particular. And its antioxidant properties as a scavenger are also reported. The aim of this study was to investigate the effect of melatonin on the in vitro maturation of mouse germinal vesicle (GV)-stage oocytes. Oocyte maturation, apoptosis, and mRNA expression of melatonin receptor were analyzed in the cumulus cell-enclosed oocytes (CEOs) cultured with melatonin for 18 h. The CEOs were obtained from 3 wk-old ICR female mice cultured in media with 0, 0.1 nM, 10 nM, or 1,000 nM melatonin for 18 h. And then the extrusion of the first polar body was assessed to evaluate the maturation rate. The apoptosis and mRNA expression of melatonin receptor (Mtnr1-a and Mtnr1-b) in cumulus cells of each group were measured by TUNEL assay, ELISA, and real time RT-PCR after in vitro maturation(IVM). The addition of melatonin in the IVM medium significantly improved nuclear maturation of the mouse GV oocytes and the highest maturation rate were obtained from the group treated with 1,000 nM melatonin. Apoptosis was not detected in IVM oocytes, but detected in cumulus cells. And cumulus cells treated with 1,000 nM melatonin exhibited significantly lower apoptosis. In the group treated with 1,000 nM melatonin, the expression of melatonin receptor mRNA was decreased in CEOs. In conclusion, melatonin has a potentially important role for regulating oocyte maturation and reduces the apoptosis of cumulus cells in vitro.

• Science of Emotion and Sensibility
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• v.14 no.4
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• pp.531-536
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• 2011

The precise measurement of human emotion is of pivotal importance in the field of emotion science. Based on the perspective of psychoneuroimmunoendocrinology, human emotion is now considered to be measurable by body fluid. The biological molecule cortisol, which is known for the stress hormone, has been widely investigated to help researchers to estimate the stress loaded on human body. Until now, several measurement techniques such as High Performance Liquid Chromatography (HPLC), fluorometric assay, and reverse phase chromatography have been developed. However, since these measurements are expensive, take relatively long time for an operation, and they are not portable, they are not appropriate for POCT (point of care testing). In this paper we demonstrate the performance of a miniaturized-microwave resonant device in the measurement of cortisol. Our method has many advantages in that it requires a small volume of sample, has fast response time, is easy to operate and needs no labeling process. Besides, it will shed a light on the measurement techniques for emotion science.

• Korean Journal of Veterinary Service
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• v.31 no.3
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• pp.397-414
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• 2008

In order to evaluate conception rate of Hanwoo in northwestern region of Gyeongsang-nam-do, we investigated conception rate and reduction of reproductive disorder rate after artificial insemination (AI) in 1,000 heads of breeding cows, This study showed that 80.9% of cows were classified as fertility after 1st and 2nd AI. For a accurate pregnancy diagnosis with practicing ovariectomy and histeotomy, we comparatively investigated each of 80 slaughtered cows, including 30 of non-pregnancy, and used enzyme-linked immunosorbent assay (ELISA) for estimation of plasma progesterone concentration and serum luteal hormone. The mean diameter of non-pregnant corpus luteum is $18.9{\pm}4.2{\times}15.6{\pm}3.6 mm$ and that of pregnant corpus luteum is $22.5{\pm}2.7{\times}18.7{\pm}2.9 mm$. This indicates that corpus luteum is more developed in the ovary of pregnant than non-pregnant cows (P<0.05). The diameter of pregnant corpus luteum according to the stage of pregnancy showed $21.3{\pm}2.4{\pm}18.4{\pm}2.6 mm$ in early stage (1-3 month), $23.4{\pm}2.8{\times}19.1{\pm}2.7 mm$ in middle stage (4-6 month) and $22.8{\pm}3.0{\times}18.8{\pm}2.4mm$, in last stage (7-9 month). This indicates that corpus luteum in middle and last stage is more significantly developed than that of early stage(P<0.05). The mean plasma progesterone concentration of cows showing size of non-pregnant corpus luteum was $4.58{\pm}0.92ng/ml$ and that of pregnant corpus luteum $8.26{\pm}0.98ng/ml$. Thus, it was more significantly increased in pregnant corpus luteum(P<0.02).. However, it was low to $0.58{\pm}0.39ng/ml$. in estrus (corpus albicans). The plasma progesterone concentration according to gestation period was high in proportion to the degree of development in corpus luteum and more significantly increased (P<0.05) and maintained in middle and last state than early state. The concentration was sharply decreased to $0.56{\pm}0.32ng/ml$ at parturition. As a consequence, we can practice the early pregnancy diagnosis by confirming non-pregnancy when the mean plasma progesterone concentration is below 1ng/ml 19 to 22 days after AI and this can be available to diagnose reproductive disorder.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.97-97
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• 2002

Human eryhropoietin (EPO) is acidic glycoprotein hormone that plays key role in hematopoiesis by facilitating differentiation of erythrocyte and formation of hemoglobin (Hb) and is used for the treatment of anemia. Human EPO is consist of 166 amino acids which is modified by three N-glycosylations (24, 38, 83) and single O-glycosylation (126). N-glycosylation is reported to be related to the cellular secretion and activity of EPO. In this study, we examined effects of mutagenesis in glycosylation site of recombinat hEPO for the cellular secretion during production from cultured CHO cell. We produced rhEpo which was cloned by PCR from human liver cDNA (TaKaRa) in cultured CHO cell. Using supernatant of the culture, ELISA assay and western analysis were performed. To estimate biological activity, 20IU of rhuEpo was subcutaneously injected into four ICR mice. After 8 days, HCT level was increased average 13 per cent, RBC was increased ca. 2${\times}$10$\^$6//${\mu}\ell$. In disease model Rat (anemia c-kit, WSRC-WS/WS), HCT was increased ca. 12%, RBC was increased ca. 1.6${\times}$10$\^$6//${\mu}\ell$. These results suggests that rhEpo we produced has biological activity. To remove glycosylation site by substituting 24, 38, 83, and 126th asparagine (or serine) with glutamic acid, overlapping -extension site-directed mutagenesis was performed. To add novel glycosylation sites, 69, 105th leucine was mutated to asparagine. Mutant EPO construct was transfected into CHO cell. Supernatant of the cell culture was analyzed using ELISA assay with monoclonal anti-EPO antibody (Medac, Germany). Since, several reports for mutagenesis of glycosylation sites showed case-by-case results, we examined both transient expression and stable expression. Addition of novel glycosylation sites resulted no secretion while deletion mutants had little effect except some double deletion mutants (24/83 and 38/83) and triple mutant. We suggest that not single but combination of glycosyl group affect secretion of EPO.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.6
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• pp.750-756
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• 2010

Two trials were conducted to study the effects of two Chinese herbal polysaccharides, Astragalus polysaccharides (APS) and Achyranthes bidentata polysaccharides (ABPS), and one Chinese herbal saponin, Acantbepanax senticosus saponin (ASS), on the immunity and growth performance of weaned pigs. Experiment 1 was a 14-day growth assay, in which 32 weaned pigs were randomly allocated to one of four dietary treatments: i) 0.05% talcum powder control; ii) 0.05% APS; iii) 0.05% mixture of APS and ASS in a 1:1 ratio by weight; and iv) 0.05% mixture of APS, ASS, and ABPS in a ratio of 1:1:1 by weight. Blood samples were collected on day 14 to determine plasma parameters. Feed intake, body weight gain, and feed efficiency were also determined. Experiment 2 was a 21-day immunity assay, in which 16 weaned pigs were randomly allotted to one of two dietary treatments: i) 0.05% talcum powder control; and ii) 0.05% mixture of APS and ASS in a 1:1 ratio by weight. On day 21, pigs were challenged with lipopolysaccharide (LPS) and 3 h later blood samples were collected and analyzed for lymphocyte proliferation as well as interleukin 6 (IL-6), insulin-like growth factor 1 (IGF-1), growth hormone (GH), and cortisol levels. In Experiment 1, feeding Chinese herbal polysaccharides and saponin increased growth performance of the pigs. The effects of the mixture of APS and ASS were especially notable, as there was a significant improvement in growth performance compared with the control (p<0.05). The plasma concentration of immunoglobulin G (IgG), nitric oxide (NO), and nitric oxide synthase (NOS) were increased in all treatments groups, with the mixture of APS and ASS increasing the level of IgG and NOS significantly (p<0.05), compared with the control. There was no difference in the NO level between the control and treatment groups (p>0.05). In Experiment 2, Chinese herbal polysaccharides and saponin showed immunostimulating effects. The level of cortisol, GH, and IGF-I were significantly increased (p>0.05), and the level of IL-6 showed a significant decrease (p<0.05) in the APS and ASS treatment after the LPS challenge. The mixture of APS and ASS could stimulate the blood lymphocyte proliferation significantly whether the LPS was injected or not (p<0.05). These results show that Chinese herbal extracts can improve growth performance and stimulate immunity of weaned pigs. A mixture of APS and ASS, compared with APS alone, could be a new kind of immunostimulant for weaned pigs, which could result in greater positive effects on their growth performance and immunity.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.36 no.2
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• pp.137-143
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• 2010

Yuza (Citrus junos Sieb ex TANAKA) is a citrus fruit that is cultivated in northeast Asia. Citron is known for containing abundant antioxidants such as vitamin C, flavonoids, for example hesperidin and hesperetin, and terpenoids such as limononin. When mature citron is processed for tea or other beverage food products in Korea, massive amounts of seeds and pericarp are remained as waste. This study aimed to exploit the processed remnant of Citron for developing functional cosmetic applications. Ethanol extracts of Yuza seed and pericarp did not show significant radical scavenging activities measured by DPPH (2,2-diphenyl-1-picrylhydrazyl) method. But they contained significantly high phenolic compounds. Cultured human dermal fibroblasts and HaCaT keratinocytes were irradiated with 25 mJ UVB and the citron extracts were added to the medium of each culture. Cellular damages caused by UVB irradiation were prevented by the addition of the Yuza extract. In addition, the reduction of the enhanced MMP-1 expression after irradiation of UVB in human dermal fibroblasts was observed. Also the increased level of pro-inflammtory TNF-$\alpha$ in the UVB irradiated HaCaT cells was decreased. The collagen expression was enhanced by the extract. Yuza extract markedly inhibited melanin production from $\alpha$-MSH treated B16F1 melanoma cells. Melanin assay, tyrosinase zymography results indicated that Yuza extract had strong depigmenting activity. In conclusion, Yuza ethanol extracts have good anti-photoaging and strong anti-melanogenic efficacies.