• Animal cells and systems
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• 제10권4호
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• pp.203-209
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• 2006

Azoles are widely used antifungal agents, which inhibit the biosynthesis of fungal cell-membrane ergosterol. In this study, using an amphibian follicle culture system, the effects of azoles on follicular steroidogenesis in frogs were examined. Itraconazole (ICZ), clotrimazole (CTZ) and ketoconazole (KCZ) suppressed pregnenolone ($P_5$) production by the follicles ($ED_{50};\;0.04_{\mu}M,\;0.33_{\mu} M,\;and\;0.91_{\mu}M$, respectively) in response to frog pituitary homogenates (FPH). However, fluconazole (FCZ), miconazole (MCZ) and econazole (ECZ) were not effective in the suppression of $P_5$ production. Not all the azoles examined suppressed the conversion of exogenous $P_5$ to progesterone ($P_4$) (by $3{\beta}$- HSD) or $P_4$ to $17{\alpha}$-hydroxyprogesterone ($17{\alpha}$-OHP) (by $17{\alpha}$-hydroxylase), or androstenedione (AD) to testosterone (T) (by $17{\beta}$-HSD). In contrast, CTZ, MCZ and ECZ in medium partially suppressed the conversion of $17{\alpha}$-OHP to AD (by C17-20 lyase) ($ED_{50};\;0.25{\mu} M,\;4.5{\mu}M,\;and\;0.7{mu}M$, respectively) and CTZ, KCZ, ECZ and MCZ strongly suppressed the conversion of exogenous T to estradiol ($E_2$) (by aromatase) ($ED_{50};\;0.02{\mu}M,\;8{\mu}M,\;0.07{\mu}M,\;0.8{\mu}M$, respectively). These results demonstrated that some azole agents strongly suppress amphibian follicular steroidogenesis and particularly, P450scc and aromatase are more sensitive to azoles than other steroidogenic enzymes.

• Animal cells and systems
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• 제11권1호
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• pp.1-8
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• 2007

Recently, we have shown that some endocrine disruptors, heavy metals, organotins and azoles suppressed steroidogenic enzymes such as P450 side-chain cleavage enzyme (P450scc) and aromatase in bullfrog ovarian follicles. In the present study, by using an amphibian ovarian follicle culture system, we examined the effects of these endocrine disruptors on maturation and ovulation of oocytes from Rana dybowskii in vitro. Ovarian fragments or isolated follicles were cultured for 24 h in a medium containing frog pituitary homogenate (FPH) or progesterone ($P_{4}$) with or without endocrine disruptors, and oocyte maturation (germinal vesicle breakdown, GVBD) and ovulation were examined. Among the organotins, tributyltin (TBT) strongly inhibited both FPH-and $P_{4}-induced$ oocyte maturation ($ED_{50}$:0.6 and 0.7 ${\mu}M$, respectively); however, tetrabutyltin (TTBT) and dibutyltin (DBT) showed only partial suppression, while monobutyltin (MBT) showed no inhibitory effect. All of the organotins suppressed $P_{4}-induced$ oocyte ovulation very effectively at a low concentration, and TBT and DBT exerted an inhibitory effect on FPH-induced ovulation. Among the heavy metals, mercury (Hg), cadmium (Cd) and cobalt (Co) were very effective in inhibiting FPH-induced oocyte maturation and ovulation, while lead (Pb), arsenite (As) and zinc (Zn) were less effective. However, all of the heavy metals suppressed FPH-induced oocyte ovulation at a high dose ($100{\mu}M$). Among the azoles, itraconazole (ICZ), ketoconazole (KCZ) and clotrimazole (CTZ) effectively inhibited FPH-induced oocyte maturation and ovulation, while econazole (ECZ), miconazole (MCZ) and fluconazole (FCZ) were considerably less effective. These results demonstrated that the abovementioned endocrine disruptors exhibited differential effects on oocyte maturation and ovulation in amphibian follicles and that the frog ovarian culture system could be used as an effective experimental tool to screen and evaluate the toxicity of various endocrine disruptors in vitro.

• Environmental Analysis Health and Toxicology
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• 제20권3호
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• pp.195-203
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• 2005

Cytochrome P45O 17$\alpha$-hydroxylase (CYPI 7) and cytorhrome P45O aromata.ie (CYPI 9) are key steroidogenic enzymes in androgen and estrogen synthesis. ThiL study evaluated the effects of nonylphenol (NP) on CYP17 and CYP19 expression in the ovary of Sprague-Dawley rats. All female rats were administered orally with the vehicle (control, corn oil), diethylstilbestrol (DES, 5.0 $\mu$g/kg) and NP (50, 100, or 200 mg/kg/day), which was startinB when they were weaned at 21 days of age for 20 days. Twenty four hours after final dose, the animals were anelthetized with ether. Significant decreases in the uterus (wet weight) were observed with 5.0 $\mu$g/kg/day DES (78$\%$, of control) and 200 mg/kg/day NP (62$\%$ of control), respectively Additionally, ovarian weight was significantly decreased with 5.0 $\mu$g/kg/day DES (63$\%$ of control) and 200 mg/kg/day NP (72$\%$ of control). The serum estradiol levels were sligHtly lower in DES and high dose NP treatment groups, but the 74 levels were not affected by DES and NP. The expression of the ovarian CYP19 gene increased with low doses (50 and 100 mg/kg/day) of NP. while DES and high dose oi NP (200 mg/kg/day) did not affect on the CYP19 mRNA levels. In contrast to the CYP19 gene, the CYP17 gene expreLsion level was significantly down-regulated by the DES and 200 mg/ks/day NP. This result suggestE that NP inhibits ovarian estrogen synthelis by supprelsing CYP17 mRNA efprelsion, And different mechanisml might exist for the expression of Lteroidogenic CYP17 and CYP19 genes in the ovary of Sprague-Dawley rats in response to NP.

• 한국발생생물학회지:발생과생식
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• 제13권3호
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• pp.163-172
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• 2009

Some organotin compounds such as butyltins and phenyltins are known to induce impo-sex in various marine animals and are considered to be endocrine disruptors. In this study, the effect of organotins on follicular steroidogenesis in amphibians was examined using ovarian follicles of Rana dybowskii and Rana catesbeiana. Isolated follicles were cultured for 6 or 18 h in the presence and absence of frog pituitary homogenate (FPH) or various steroid precursors, and the levels of product steroids in the culture media oassay. Among the butyltin compounds, tributyltin (TBT) strongly and dose-dependently inhibited the FPH-induced synthesis of pregnenolone ($P_5$) and progesterone ($P_4$) by the follicles. TBT also strongly suppressed the conversion of cholesterol to $P_5$ and partially suppressed the conversion of $P_5$ to $P_4$. A high concentration of dibutyltin (DBT) also inhibited steroidogenesis by the follicles while monobutyltin and tetrabutyltin had negligible effects. The toxic effect of TBT or DBT was irreversible and a short time of exposure (30 min) was enough to suppress steroidogenesis. All the phenyltin compounds significantly inhibited FPH-induced $P_5$ synthesis by the follicles. The effective dose of 50% inhibition by diphenyltin was $0.04\;{\mu}M$ and those of monophenyltin and triphenyltin were $0.24\;{\mu}M$ and $0.3\;{\mu}M$, respectively. However, none of the phenyltin compounds significantly suppressed the conversion of $P_4$ to $17{\alpha}$-hydroxyprogesterone ($17{\alpha}$-OHP) (by $17{\alpha}$-hydroxylase), $17{\alpha}$-OHP to androstenedione (AD) (by $C_{17-20}$ lyase), or AD to testosterone by the follicles. Taken together, the data show that among the steroidogenic enzymes, P450scc in the follicles is the most sensitive to organotin compounds and that an amphibian follicle culture system can be a useful screening model for endocrine disruptors.

• 생명과학회지
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• 제24권10호
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• pp.1039-1045
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• 2014

본 연구의 목적은 마우스 레이디히 세포인 mLTC-1 세포에 사람 융모성 성선자극호르몬인 hCG를 처리하여 유도되는 소포체 스트레스가 IRE1/XBP1 경로를 통하는지 분석하는 것 이다. 이전 연구에서 hCG처리에 의해 레이디히 세포는 소포체 스트레스 매개의 세포자멸사가 유도될 뿐만 아니라 ATF6경로를 조절함으로써 UPR이 성호르몬 합성효소의 발현에 중요한 역할을 하는 것을 증명하였다. UPR 경로는 또한 IRE1/XBP1 경로를 통하여 조절되는 것이 알려져 있지만 레이디히 세포에서 hCG에 의한 소포체 스트레스에 의해 IRE1/XBP1 경로의 활성화가 유도되는지에 대해서는 거의 알려져 있지 않다. mLTC-1세포에서 hCG 처리 후 IRE1/XBP1경로의 활성을 조사하기 위하여, 인산화된 IRE1 단백질 확인하기 위한 western blot, XBP1 mRNA splicing 확인하기 위한 RT-PCR을 수행하였다. 또한 우리는 IRE의 활성을 관찰하기 위하여 소포체 스트레스-활성 표지자(ERAI) construct를 이용하고 이를 형광현미경과 flow cytometry를 이용하여 분석하였다. 결과적으로, hCG 처리에 의해 인산화된 IRE1 단백질의 발현 수준이 두드러지게 증가하였다. F-XBP1-venus/F-$XBP1{\Delta}DBD$-venus가 도입된 mLTC-1 세포에서, hCG 처리에 의해 녹색 형광을 띄는 세포들이 유도되었고 각각 핵/세포질에서 발현하는 것을 확인할 수 있었다. 게다가 hCG 처리 후에 XBP1 mRNA의 splicing 또한 상당히 증가되는 것을 확인 할 수 있었다. 이 결과들을 통하여 종합해 볼 때 레이디히 세포에서 hCG 처리에 의해 유도되는 소포체 스트레스가 IRE1/XBP1 경로의 활성을 유발하는 것을 확인 할 수 있었다.