• 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.

• The Korean Journal of Physiology and Pharmacology
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• v.9 no.4
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• pp.231-238
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• 2005

Neuronal apoptotic events, which result in cell death, are occurred in hypoxic/ischemic conditions. Estradiol is a female sex hormone with steroid structure known to provide neuroprotection through multiple mechanisms in the central nervous system. This study was aimed to investigate the signal transduction pathway of $CoCl_2$-induced neuronal cell death and the inhibitory effects of estradiol. Administration of $CoCl_2$ decreased cell viability in both a dose- and time-dependent manner in PC12 cells. $CoCl_2$-induced cell death produced genomic DNA fragmentation and morphologic changes such as cell shrinkage and condensed nuclei. It was found that $CoCl_2$-treated cells increased the reactive oxygen species (ROS) as well as caspase-8, -9 and -3 activities. However, pretreatment with estradiol before exposure to $CoCl_2$ prevented the reduction in cell viability reduction and attenuated DNA fragmentation and morphologic changes caused by $CoCl_2$. Furthermore, the $CoCl_2$-induced increases of ROS levels and caspases activities were attenuated by estradiol. Gene expression analysis revealed that estradiol blocked the underexpression of the Bcl-2 and ameliorated the increase in the release of cytochrome c from mitochondria into cytoplasm and Fas-ligand (Fas-L) upregulated by $CoCl_2$. These results suggest that $CoCl_2$ induce apoptosis in PC12 cells through both mitochondria- and death receptor-mediated cell death pathway. Estradiol was found to have a neuroprotective effect against $CoCl_2$-induced apoptosis through the inhibition of ROS production and by modulating apoptotic effectors associated with the mitochondria- and death-dependent pathway in PC12 cells.

• Korean Journal of Animal Reproduction
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• v.11 no.1
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• pp.73-84
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• 1987

This experiment was carried out to investigate the effects of hormone addition(FSH, HCG, estrogen and progesterone) and composition (BSA and FCS) of mKRB on the in vitro maturation and fertilizability of follicular oocytes of the Korean native cattle. The ovaries were removed at a slaughterhouse, returned to laboratory in a thermostat (30-35$^{\circ}C$) within 4 hr, and collected by aspirating normal follicles which had diameters of 1 to 6 mm. The oocytes with cumulus cells were cultured for 8, 16, 24 and 30 hr in a modified KRB solution containing BSA or FCS and hormones. The in vitro matured oocytes in mKRB containing FCS, FSH and steroids were transferred in the rabbit uterus for examination of their in vivo fertilizability with bovine sperm preincubated 4 to 6 hr in the rabbit uterus. 1. The mean number of oocytes collected per cattle was 6.5 from 1-3mm follicles, 1.3 from 4-6mm follicles, and total was 7.7. 2. The meiotic division at 16hr-cuture in the oocytes from 1-3mm follicles was slightly stimulated by the addition of FSH in mKRB + BSA solution compared with the control. At 30hr-culture, their maturation rates(%Met II) were also increased by FSH of 1 $\mu\textrm{g}$/ml(38.4%) and 5$\mu\textrm{g}$/ml(35.7%) as compared with the control (21.4%). The maturation rate at 30hr-culture in the oocytes from 4-6mm follicles was 53.8% and 57.1% by the FSH addition of 1$\mu\textrm{g}$/ml and 5$\mu\textrm{g}$/ml, respectively. These rates were similar with the control(57.1%), but higher than those of oocytes from 1-3mm follicles. 3. The meiotic division at 16hr-culture in the oocytes from 1-3mm follicles was stimulated by the HCG addition of 1IU/ml and 5IU/ml. However, the maturation rate at 30hr-culture was greatly decreased by the HCG addtion (26.6% and 13.3%) compared with the control(53.3%) and these rates (30.8%) in the oocytes from 4-6mm follicles were also lower than that fo the control(58.3%). 4. Low maturation rate (37.5%) of the oocytes cultured in mKRB containing BSA and 5IU/ml HCG was increased (55.0%) when 15% FCS with HCG was added to mKRB instead of BSA. 5. When 16hr-cultured oocytes in mKRB containing BSA and gonadotropins (5$\mu\textrm{g}$/ml FSH and 5IU/ml HCG) were transferred in the medium without gonadotropins and recultured for 16hr, the maturation rate of HCG-treated oocytes was greatly improved. 6. The maturation rates of oocytes were greatly affected by steroids. The combined addition of FCS+FSH+estrogen or +progesterone to mKRB increased the maturation rate compared with the combination of BSA+FSH or FCS+FSH in mKRB. 7. The fertilization rate, presence of pronuclei, was increased by the combination of FCS+FSH+p in mKRB as compared with that (5.6%) of BSA+FSH and the rates of FCS+FSH+steroids ranged from 12.5 to 17.6%.

• Development and Reproduction
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• v.7 no.1
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• pp.41-48
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• 2003

This report aimed at investigating the differential gene expressions of the adhesion receptors between ovariectomized (OVX) and estrus stage rat uteri (OVX vs. estrus pair) using the cDNA expression away analysis. In addition, this report aimed at confirming of the differential gene expressions of the adhesion receptors between OVX and progesterone (P$_4$) injected OVX rat uteri (OVX vs. OVX+P$_4$ pair). RNA samples were extracted from the uterus and reverse-transcribed in the presence of [$\alpha$$^{32}$ P]-dATP. Membrane sets of Rat Atlas array 1.2 II (Clontech) were hybridized with CDNA probe sets. RT-PCR was employed to validate the relative gene expression patterns obtained by the cDNA array. The results were well consistent to cDNA array analysis data except the fold changes of gene expression. Among a total of 1176 cDNAs, 5 genes of adhesion receotor including embigin protein, activated leukocyte cell adhesion molecule, afadin, neuroligin 2, semaphorin Z showed significant (more than 2-fold) changes in the OVX vs. late estrus pair. All of these genes were up regulated in estrus stage than OVX rat uterus. In the OVX vs. OVX+P$_4$ pair, 4 genes including osteonectin, afadin, neuroligin 2, semaphorin Z showed significant changes. All of these genes were also up regulated in OVX+P$_4$ injected rat uterus than OVX control. Three genes including afadin, neuroligin 2, semaphorin Z which were up regulated in estrus and OVX+P$_4$ injected rat uteri of both experimental pairs than OVX rat uteri. These genes seem to be under the control of P$_4$.

• Development and Reproduction
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• v.17 no.3
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• pp.269-274
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• 2013

This study was performed to evaluate the effects of fibroblast co-culture on in vitro maturation (IVM) of prepubertal mouse preantral follicles. The intact preantral follicles were obtained from the ovaries of 12-14 day old mice and these were cultured individually in ${\alpha}$-minimal essential medium (${\alpha}$-MEM) supplemented with 5% fetal bovine serum (FBS), $100mIU/m{\ell}$ recombinant follicle stimulating hormone (rFSH), 1% insulin-transferrin-selenium, $100{\mu}g/ml$ penicillin and $50{\mu}g/m{\ell}$ streptomycin as base medium for 12 days. A total of 200 follicles were cultured in base medium co-cultured with mouse embryonic fibroblast (MEF) (MEF group) (n=100) or only base medium as control group (n=100). Survival rate of follicles on day 12 of culture were significantly higher in the MEF group of 90.0%, compared with 77.0% of the control group (p=0.021). Follicle diameters on day 6 and 8 of the culture period were significantly larger in the MEF group than those in the control group (p=0.021, p=0.007, respectively). Estradiol levels in culture media on day 4, 6, 8, 10 and 12 of the culture period were significantly higher in the MEF group (p=0.043, p=0.021, p=0.006, p<0.001 and p=0.008, retrospectively). Our data suggest that MEF cell co-culture on IVM of mouse preantral follicle increases survival rate and promotes follicular growth and steroid production.

• Korean Journal of Animal Reproduction
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• v.18 no.2
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• pp.141-150
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• 1994

Morphological features of the interaction between the hatching blastocyst and implantation in pig were studied by electron microscopy. The observations extended from late blastocyst stage to the completion of trophoblastic erosion of the epithelium and early decidual transformation of the epithelium and early decidual transformation of the stromal cells. Between day 7 and 17 of pregnancy, blastocysts from 0.3 to 12 mm in diameter were flushed from the uterine horns of Dutch Landrace pigs. On the 7th of development in the pig blastocyst, the blastocyst shedded of the zona pellucida established the tips of microvilli and with bleb-like cytoplasmic protrusions of the epithelial cells. From day 11 on in pig embryo, the bilayered trophoblast undergoes a dramatic phase of elongation so that the initially spherical expanded blastocyst becomes tubular. In pig, close apposition to the uterine wall beg-ins at about 12 $^1$/$_2$ days and then attachment occurred during the afternoon of the 16th or 18th day post coitum. At this stage, embryonic loss compared with corpus luteum number is up to 40% of ovulated oocytes. Therefore, the implantation failture of these embryos may be mainly caused by morphological abnormality and failture of zona shedding.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.45 no.3
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• pp.253-261
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• 2012

We investigated the reproductive capacity of starry flounder Platichthys stellatus from the Uljin (UJ) marine ranching area and compared it to that of P. stellatus from the coastal waters of Pohang (PH). In UJ, female gonadosomatic index (GSI) peaked in October ($3.14{\pm}0.87$) and male GSI was high in October and December. In PH, female GSI peaked in January ($18.64{\pm}2.15$) while male GSI began to increase in October and remained high until March. Most ovaries of UJ females were immature with perinucleus oocytes, although the testes of UJ males were ripe in January. Both the ovaries and testes of PH starry flounders were ripe from January to March. The plasma estradiol-$17{\beta}$ ($E_2$) levels of UJ females were highest in October ($4.09{\pm}1.90$ ng/mL) although the testosterone (T) levels of UJ males were highest in December ($3.81{\pm}0.78$ ng/mL) and decreased gradually until April. The $17{\alpha}$,$20{\beta}$-dihydroxy-4-pregnen-3-one ($17{\alpha}20{\beta}P$) levels of UJ females were not detected. The E2 levels of PH females were highest in December ($36.25{\pm}33.07$ ng/mL) and $17{\alpha}20{\beta}P$ levels were highest in March ($5.51{\pm}0.95$5 ng/mL). The T levels of PH males were highest in December ($4.03{\pm}1.34$ ng/mL) and decreased gradually until October. Taken together, these results suggest that most females from UJ did not reach maturation with a spawning period that was considered to be between December and January.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.149-156
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• 1999

Steroid hormone is known to cause the dynamic changes of mammalian uterus during reproductive cycle, which are modulated via hypothalamus-pituitary -gonad reproductive endocrine axis. Although there were so many studies about estrogenic regulation of uterine growth and differentiation. There is little information about the effect of estrogen on the expression of various transcription factors involved in gene expression. Thus the present study was designed to demonstrate E induced expression of c-fos, c-jun, hsp25 mRNA in rat uterus. Employing Northern blot analysis, we studied the temporal expressions of c-fos, c-jun, and hsp25 messenger RNAs (mRNAs) elicited by a single 17beta-estradiol (E) treatment in the uteri of bilaterally ovariectomized adult rats. c-fos, c-jun, and hsp25 mRNA levels were increased and peaked at 3h after E administration, and then c-fos and c-jun mRNA levels were rapidly decreased to basal control level while, increased hsp25 mRNA levels were sustained till 12h post E treatment. To test the estrogenic effect on the increase of c-fos, c-jun, and hsp25 mRNA levels, we also examined the effects of antiestrogen (tamoxifen). Pretreatment with tamoxifen effectively blocked the E-induced increase of c-fos, c-jun, and hsp25 mRNA levels at 3h post E treatment. Present results suggest that transient increase of c-fos and c-jun protooncogene mRNA at the early time and simultaneous expression of hsp25 mRNA contribute to the response of uterine tissues to E in adult female rats.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.4
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• pp.463-470
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• 2011

Gonadotropin-releasing hormone receptor (GnRHR) gene is expressed at the anterior pituitary gland and plays a key role in gonad development. This study aimed to investigate molecular genetic characteristics of the GnRHR gene and elucidate the effects of single nucleotide polymorphisms (SNPs) of GnRHR gene on sex steroid level in Japanese flounder (Paralichthys olivaceus). We used polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and sequencing of the GnRHR gene in 75 individuals. We identified three SNPs in the GnRHR gene: P1 locus (C759A and C830T) in the coding region of exon2 which were both linked together and P2 locus (G984T) in the coding region of exon3, which added a new transcript factor (ADR1) and a new methylation site (CG). Only C830T of P1 leads to amino acid changes Thr266Ile. Statistical analysis showed that P1 was significantly associated with $17{\beta}$-estradiol ($E_2$) level (p<0.01) and gonadosomatic index (GSI) (p<0.05). Individuals with genotype BB of P1 had significantly higher serum $E_2$ levels (p<0.01) and GSI (p<0.05) than those of genotype AA or AB. Another SNP, P2, synonymous mutation, was significantly associated with GSI (p<0.05). Individuals with genotype AB of P2 had significantly higher GSI (p<0.05) than that of genotype AA. In addition, there was a significant association between one diplotype based on three SNPs and reproductive traits. The genetic effects for both serum $E_2$ level and GSI of diplotype D4 were super diplotypes (p<0.05). These results suggest that the SNPs in Japanese Flounder GnRHR are associated with $E_2$ level and GSI.

• Journal of Plant Biotechnology
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• v.44 no.3
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• pp.264-270
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• 2017

Brassinosteroid (BR), a plant steroid hormone, plays a critical role in the growth and developmental processes through its canonical signaling and crosstalk with various internal and external signaling pathways. Recent studies have revealed the essential interplay mechanisms between BR and ABA during seed germination and early seedling establishment. However, molecular mechanisms for this important signaling crosstalk are largely unknown. To understand the crosstalk between BR-mediated signaling pathways and ABA functions during early seedling development, we carried out a comparative genome-wide transcriptome analysis with an Agilent Arabidopsis $4{\times}44K$ oligo chip. We selected and compared the expression patterns of ABA response genes in ABA-insensitive bes1-D mutant with wild type seedlings on which ABA was exogenously applied. As a result, we identified 2,353 significant differentially expressed genes (DEGs) in ABA-treated bes1-D and wild type seedlings. GO enrichment analysis revealed that ABA signaling, response, and metabolism were critically down-regulated by BR-activated signaling pathways. In addition, the genome-wide transcriptome analysis data revealed that BR-regulated signaling pathways were tightly connected to diverse signal cues including abiotic/biotic stress, auxin, ROS etc. In this study, we newly identified the molecular mechanisms of BR-mediated repression of ABA signaling outputs. Also, our data suggest that interplay among diverse signaling pathways is critical for the adaptive response of the plant to various environmental factors.