• Clinical and Experimental Reproductive Medicine
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• 제47권3호
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• pp.194-206
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• 2020

Objective: The aim of this study was to investigate microRNAs (miRNAs) related to follicle-stimulating hormone (FSH) responsiveness using miRNA microarrays and to identify their target genes to determine the molecular regulatory pathways involved in FSH signaling in KGN cells. Methods: To change the cellular responsiveness to FSH, KGN cells were treated with FSH receptor (FSHR)-specific small interfering RNA (siRNA) followed by FSH. miRNA expression profiles were determined through miRNA microarray analysis. Potential target genes of selected miRNAs were predicted using bioinformatics tools, and their regulatory function was confirmed in KGN cells. Results: We found that six miRNAs (miR-1261, miR-130a-3p, miR-329-3p, miR-185-5p, miR-144-5p and miR-4463) were differentially expressed after FSHR siRNA treatment in KGN cells. Through a bioinformatics analysis, we showed that these miRNAs were predicted to regulate a large number of genes, which we narrowed down to cytochrome P450 family 19 subfamily A member 1 (CYP19A1) and estrogen receptor alpha (ESR1) as the main targets for miR-4463. Functional analysis revealed that miR-4463 is a regulatory factor for aromatase expression and function in KGN cells. Conclusion: In this study, we identified differentially expressed miRNAs related to FSH responsiveness. In particular, upregulation of miR-4463 expression by FSHR deficiency in human granulosa cells impaired 17β-estradiol synthesis by targeting CYP19A1 and ESR1. Therefore, our data might provide novel candidates for molecular biomarkers for use in research into poor responders.

• Asian Pacific Journal of Cancer Prevention
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• 제14권5호
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• pp.2819-2822
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• 2013

In this study, antiproliferative effects of the selective estrogen receptor modulator Tamoxifen and the aromatase inhibitor letrozole (Femara) were evaluated and compared using the FM3A cell line, originating from a C3H mouse mammary carcinoma and positive in terms of estrogen receptor (ER) expression. Cell kinetic parameters including labelling index, mitotic index and labelling index were assessed after exposure of the. FM3A cell line to $0.001{\mu}g/ml$ of Tamoxifen and $0.25{\mu}g/ml$ of Femara for 4, 8, 16 and 32 h for all parameters. The results showed that cell growth was inhibited by both agents. There was a significant decrease in labelling index and mitotic index and significant increase in apoptotic index for all experimental groups. The differences between control and all experimental groups were statistically significant (p<0.001) for all applications.

• Asian-Australasian Journal of Animal Sciences
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• 제31권5호
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• pp.658-671
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• 2018

Objective: Nandrolone decanoate (ND) is an anabolic-androgenic steroid frequently used for clinical treatment. However, the inappropriate use of ND results in the reduction of serum testosterone level and sperm production. The suppressive effect of ND on testosterone production has not been investigated in detail. The present study was designed to examine the effect of ND on the expression of steroidogenic enzymes in the rat testis. Methods: Male Sprague Dawley rats at 50 days of age were subcutaneously administrated with either 2 or 10 mg of ND/kg body weight/week for 2 or 12 weeks. The changes of transcript and protein levels of steroidogenic enzymes in the testis were determined by real-time polymerase chain reaction and western blotting analyses, respectively. Moreover, immunohistochemical analysis was employed to determine the changes of immunostaining intensity of these enzymes. The steroidogenic enzymes investigated were steroidogenic acute regulatory protein, cytochrome P450 side chain cleavage enzyme, $17{\alpha}-hydroxylase$, $3{\beta}-hydroxysteroid$ dehydrogenase, and cytochrome P450 aromatase. Results: The treatment of ND resulted in depletion of Leydig cells and sloughing of germ cells in the testis. The ND treatment caused significant expressional decreases of steroidogenic enzymes at transcript and protein levels, and the destructive effects of ND on the testis were more apparent with a higher dose and a longer period of the treatment. Evident reduction of immunostaining intensity present in Leydig cells was clearly detected by the ND treatment. Conclusion: The exposure to ND in young male results not only in histological changes of the testis but also in aberrant gene expression of testicular steroidogenic enzymes, consequently leading into the reduction of testosterone production in the testis and thus likely disruption of spermatogenesis.

• Clinical and Experimental Reproductive Medicine
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• 제34권4호
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• pp.275-283
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• 2007

목 적: 본 연구에서는 생리혈에 존재하는 탈락된 자궁내막조직에서의 자궁내막증 관련 유전자들의 발현 양상과 자궁내막증 병태생리와의 관련성을 살펴보고자 하였다. 연구방법: 자궁내막증으로 확진된 환자 (n=16)와 정상 대조군 (n=26)에서 생리주기 2$\sim$3일째 Wallace catheter로 채취한 생리혈로부터 탈락된 자궁내막조직을 분리하였다. 기존의 연구들에서 보고된 12종류의 자궁내막증 관련 유전자들의 mRNA 발현 양상을 semi-quantitative RT-PCR 방법으로 비교, 분석하였다. 결 과: 생리혈에서 분리한 탈락된 자궁내막조직은 조직학적 관찰을 통해 자궁내막조직임을 확인하였다. 총 12가지 종류의 자궁내막증 관련 유전자에 대한 RT-PCR 분석에서 telomerase, c-kit, aromatase등의 mRNA 발현이 관찰되지 않았다. 세포사멸 (apoptosis)과 관련성이 있는 fas, fas ligand, bcl-2, bax 유전자와 stem cell factor, ER-$\alpha$/$\beta$, endometriosis protein-I, secretory leukocyte protease inhibitor 등의 mRNA 발현 양상은 자궁내막증으로 확진된 환자군과 대조군에서 통계적으로 유의한 차이를 나타내지 않았다. 결 론: 결론적으로 자궁내막증과 관련된 다양한 유전자들의 발현 양상을 생리혈에 존재하는 탈락된 자궁내막조직에서 분석하였지만 의미성이 있는 유전자를 동정하지는 못하였다. 이는 자궁내막조직의 생리학적 특징인 생리주기에 따른 유전자 발현의 역동적인 변화와 관련이 있을 것으로 생각된다.

• Journal of Animal Science and Technology
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• 제63권6호
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• pp.1247-1264
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• 2021

Anabolic steroids are frequently used to increase the growth rate of meat-producing animals. Exposure to an anabolic-androgenic steroid, nandrolone decanoate (ND), is associated with expressional reduction of testicular steroidogenic enzymes. However, the effect of withdrawal of ND exposure on the expression of these testicular molecules has not been thoroughly explored. The current research investigated expression changes of testicular steroidogenic enzymes in rats at several recovery periods (2, 6, and 12 weeks) after the stop of ND treatment with different doses (2 and 10 mg/kg body weight) for 12 weeks. Body and testis weights were recorded, and transcript levels of molecules were determined by quantitative real-time polymerase chain reaction (PCR). The immunohistochemistry was used to examine the changes of immuno-intensities of molecules. At 6 and 12 weeks of the recovery period, the 10 mg/kg ND-treated rats were lighter than other experimental groups. The interstitial compartment vanished by ND treatment filled up as the recovery period became longer. The expression of steroidogenic acute regulatory protein was returned to the control level at 12 weeks of the recovery period. Expression levels of cytochrome P450 side-chain cleavage and 17a-hydroxylase were increased in 2 mg/kg ND-treated group at 6 weeks of the recovery period, and transcript levels of these molecules in 2 and 10 mg/kg ND-treated groups at 12 weeks of the recovery period were significantly lower than the control. Expression levels of 3β-hydroxysteroid dehydrogenase (HSD) type I and 17β-HSD type 3 in 2 mg/kg ND-treated group were comparable with those of control at 12 weeks of the recovery period, but not in 10 mg/kg ND-treated group. Expression of cytochrome P450 aromatase (Cyp19) was reverted to the control level at 2 weeks of the recovery period. Except for Cyp19, there was a visible increase of immuno-staining intensity of other testicular steroidogenic enzymes in the Leydig cells as the recovery period progressed. This research has demonstrated that the cease of ND administration could restore the expression of testicular steroidogenic enzymes close to the normal level. Nevertheless, a relatively long recovery period, compared to the ND-exposure period would be required to retrieve normal expression levels of testicular steroidogenic enzymes.

• Asian-Australasian Journal of Animal Sciences
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• 제27권12호
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• pp.1695-1704
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• 2014

Maternal malnutrition during pregnancy may give rise to female offspring with disrupted ovary functions in adult age. Neonatal ovary development predisposes adult ovary function, yet the effect of maternal nutrition on the neonatal ovary has not been described. Therefore, here we show the impact of maternal protein restriction on the expression of folliculogenic and steroidogenic genes, their regulatory microRNAs and promoter DNA methylation in the ovary of neonatal piglets. Sows were fed either standard-protein (SP, 15% crude protein) or low-protein (LP, 7.5% crude protein) diets throughout gestation. Female piglets born to LP sows showed significantly decreased ovary weight relative to body weight (p<0.05) at birth, which was accompanied with an increased serum estradiol level (p<0.05). The LP piglets demonstrated higher ratio of bcl-2 associated X protein/B cell lymphoma/leukemia-2 mRNA (p<0.01), which was associated with up-regulated mRNA expression of bone morphogenic protein 4 (BMP4) (p<0.05) and proliferating cell nuclear antigen (PCNA) (p<0.05). The steroidogenic gene, cytochrome P450 aromatase (CYP19A1) was significantly down-regulated (p<0.05) in LP piglets. The alterations in ovarian gene expression were associated with a significant down-regulation of follicle-stimulating hormone receptor mRNA expression (p<0.05) in LP piglets. Moreover, three microRNAs, including miR-423-5p targeting both CYP19A1 and PCNA, miR-378 targeting CYP19A1 and miR-210 targeting BMP4, were significantly down-regulated (p<0.05) in the ovary of LP piglets. These results suggest that microRNAs are involved in mediating the effect of maternal protein restriction on ovarian function through regulating the expression of folliculogenic and steroidogenic genes in newborn piglets.

• Journal of Applied Biological Chemistry
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• 제59권4호
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• pp.305-311
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• 2016

본 연구는 야관문과 호로파 복합추출물인 YHM이 과산화수소($H_2O_2$)로 산화적스트레스를 가한 TM3 세포의 테스토스테론 발현에 미치는 영향을 확인하고자 수행되었다. 세포독성 시험을 수행하여 YHM의 경우 $40{\mu}g/mL$을 최고 농도로 중농도 $20{\mu}g/mL$, 저농도 $10{\mu}g/mL$ 처리군을 설정하였고, TM3 세포에 산화적 스트레스를 주기 위해서는, serum free 배지에 $50{\mu}M$의 과산화수소를 4시간 동안 처리하였다. 산화적 스트레스가 가해진 TM3 세포에 YHM 시료를 처리하여 세포 생존률에 미치는 영향을 평가하였을 때 모든 농도 처리군에서 세포증식이나 독성이 없었다. 테스토스테론은 과산화수소를 처리하였을 때 감소하였다가 YHM 시료를 처리하였을 때 control 수준으로 회복되거나, control 보다 더 증가하였다. 또한 시료에 의한 테스토스테론 양의 증가원인을 확인하기 위하여, 테스토스테론 합성 및 분해에 관여하는 효소들의 발현량을 ELISA와 Real-time PCR을 통해 알아보았다. 테스토스테론 합성에 관여하는 $3{\beta}$-HSD4와 17,20-desmorase는 과산화수소 처리 시 감소하였다가, YHM을 처리하였을 때는 control 수준으로 회복하였다. 테스토스테론을 estradiol 및 dihydrotestosterone로 변환시키는 aromatase와 $5{\alpha}$-reductase2는 과산화수소를 처리하였을 때 증가하였다가 YHM 시료를 처리하면 control 수준이나 그 이하로 감소하였다. 이 결과들로 보았을 때 YHM 시료는 TM3 세포의 증식에는 영향을 미치지 못 하지만, 산화적 스트레스에 의해 감소된 테스토스테론 합성 효소의 발현을 증가시키고, 반대로 증가되는 테스토스테론 분해 효소의 발현은 감소시켜, 결국 산화적 스트레스에 의해 저하된 테스토스테론의 양을 회복시키거나 증가시키는 효과가 있는 것으로 판단된다.

• 한국환경보건학회지
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• 제41권3호
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• pp.147-162
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• 2015

Objectives: The objective of this review was to summarize the primary role of three representative endocrine axes in aquatic vertebrates and discuss the effects on endocrine systems and their interactions in teleost fish after exposure to environmental contaminants. Methods: We summarized individual traits and mechanisms for hormonal and transcriptional interactions between the hypothalamic-pituitary-gonad (HPG), hypothalamic-pituitary-thyroid (HPT), and hypothalamic-pituitary-adrenal (HPA) axes in fish. We also provided a brief discussion on the effects of nonylphenol-induced toxicity on endocrine systems and their interactions in fish as a demonstration of holistic explanation. Results: Currently-available data showed that thyroid dysfunction is associated with reproductive toxicity due to changes in steroidogenic gene expressions and sex hormone levels as well as gonad glands in fish. As an example, we demonstrated that exposure to nonylphenol could induce estrogenicity in male fish by decreasing thyroid hormones, which contributes to increased aromatase expression. Although the mechanisms are complicated and involved in multiple ways, a number of studies have shown that sex steroids influence the HPT axis or the HPA axis in fish, indicating bi-directional crosstalk. Critically missing is information on the primary target or toxicity mechanisms of environmental contaminants among the three endocrine axes, so further studies are needed to explore those possibilities. Conclusions: This review highlights the interactions between the HPG, HPT, and HPA axes in fish in order to better understand how these endocrine systems could interact with each other in situations of exposure to endocrine disrupting chemicals.

• 생명과학회지
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• 제28권2호
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• pp.265-273
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• 2018

에스트로겐(E2)은 유방암의 발달과 진행에 관여하며, 에스트로겐 수용체(ER)에 의해 매개된다. ER은 유방암세포에서 epidermal growth factor receptor와 insulin-like growth factor-1 receptor의 신호전달경로들 사이에서 다양한 cross-talk을 통하여 세포의 증식, 이주, 침습 및 약물에 대한 저항성을 일으키는데 중요한 역할을 수행한다. 유방암은 내분비신호전달의 항상성 붕괴에 의해 주로 발생되며, 특히 E2/IGF-1/EGF와 ER/G-protein estrogen receptor (GPER)/IGF-1R/EGFR, 그리고 이들의 세포내 신호전달 매개인자들의 통제되지 않는 발현과 활성증가에 의해 유발된다. 이러한 변화는 E2와 성장인자 신호전달 사이의 복잡한 cross-talk에 영향을 주어 결국 암의 진행과 내분비조절인자들에 대한 저항성을 갖게 된다. 따라서, E2와 성장인자들 사이의 cross-talk에 관한 분자적 기전을 단계별로 규명하는 것은 유방암의 다양한 유형에 따른 맞춤형 치료에 기여할 것으로 사료된다. 특히, 다양한 유전형 및 표현형을 가진 유방암의 치료를 위한 전략으로서, ER+ 호르몬의존성 유방암세포에 대한 aromatase 억제제 및 E2작용 차단제의 사용과 E2와 성장인자들 사이의 cross-talk에 의한 암세포의 증식억제를 위한 IGF-1R/EGFR 활성차단제의 사용 등을 들 수 있다. 뿐만 아니라, ER과 EGFR/IGF-1R 사이의 cross-talk에 의해 조절되는 ECM 분자들의 발현변화는 유방암세포의 전이에 대한 표적치료제를 위해 활용될 수 있다. 따라서, 암의 진행과 관련된 ER, GPER, IGF-1R 및 EGFR 매개에 의한 신호전달경로들 사이의 cross-talk에 관한 보다 더 자세한 분자적 수준의 규명이 필요할 것으로 사료된다.

• Clinical and Experimental Reproductive Medicine
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• 제33권1호
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• pp.15-23
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• 2006

목 적: 본 연구는 흰쥐 난소를 실험모델로 하여 미성숙 전동 난포의 성장에 대한 pituitary adenylate cyclase-activating polypeptide (PACAP)의 영향을 얄아보고자 하였다. 연구방법: 미성숙 전동 난포를 생후 21일된 흰쥐로부터 분리하여 PACAP을 첨가하거나 첨가하지 않은 무혈청 배양액에서 3일 동안 배양하고, 푸로게스테론 호르몬의 생성, 난포의 성장, 과립막세포의 증식 및 유전자의 동태 등을 관찰하였다. 증식의 정도는 thymidine incorporation 방법으로 검색하고 유전자의 변동은 Northern 분석을 이용하였다. 결 과: PACAP으로 처리한 군은 난포의 직경이 75% 증가한 반면 난포자극호르몬인 FSH로 처리한 군은 65% 증가하였고, PACAP 처리는 과립막 세포의 증식을 강화시켰다. FSH와 PACAP 공히 배양된 흰쥐 난포의 과립막 세포와 FSH에 반응하는 세포주인 GFSHR-17에서의 프로게스테론 생성을 촉진시켰고, PACAP이 FSH의 작용을 증진시켜 SF-1과 아로마타제 유전자 발현을 촉진시켰다. 결 론: 본 연구는 PACAP이 과립막증식과 스테로이드합성을 통하여 전동 난포의 성장을 촉진함을 시사하였고, 또한, SF-1, 아로마타제 등에 대한 FSH의 작용을 도와주는 역할을 PACAP이 담당하므로 PACAP은 초기 난포성장에 필요한 난소국소인자임을 유추할 수 있었다.