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화학적 요법을 이용한 넙치의 성 성숙 억제 기술 개발에 관한 연구

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Alternative Title
Development of sexual maturation inhibition technique by using chemical method in olive flounder (Paralichthys olivaceus)
Sex determination in some reptiles, amphibians, and fishes is known to be influenced greatly by environmental factors. Successful reproduction of vertebrates necessitates complex interactions along the brain-pituitary-gonad axis, it is determined by gonadotropin releasing hormone produced in the hypothalamus of the brain, gonadotropin synthesized in the pituitary gland, and sex hormone secreted by the gonads. The activity of the Estrogen (E2) is regulated through the Estrogen receptor (ER), which is divided into two subtypes, alpha and beta. In fish, the ER has been reported to be expressed in the brain, gonads, and liver. Vitellogenin (VTG) is a sex-specific protein that appears in the blood of mature females of oviparous vertebrates. The E2 secreted from eggs binds to the ER in hepatocytes to synthesize VTG proteins. It is known that VTG receptors are produced in eggs, and that ligands are produced in the liver. It is known that if a large amount of aromatase is secreted, the sex is changed to female, and if a small amount is secreted by an exogenous treatments of Fadrozole, it is changed to male.
Dopamine (Da) is a neurotransmitter that plays an important role in reproductive function, and plays a decisive role in inducing the breeding cycle in seasonal breeding animals, especially by regulating changes in gonadotropin. The sex hormones in fish include Gonadotropin releasing hormone (GnRH), Gonadotropic hormone (Gth), E2, etc.
Aromatase inhibitor (AI) blocks P450arom activity to inhibit aromatase enzyme activity and inactivates E2 production from androgen. In humans, ovarian aromatase in premenopausal women is responsible for a significant amount of circulating E2 and is extremely sensitive to changes in luteinizing hormone (LH) ; AI is also used to treat cancer in women. Drugs that function as AIs include Anastrozole (Ana), Exemestane (Exe), Fadrozole, Letrozole and Vorozole, all of which inhibit the production of E2 from androgen by inhibiting aromatase activity. Researchers have investigated AI in various gonochoristic fish species and sex changing protogynous species, including fish, to induce female virilization.
Tamoxifen (Tam), an anti-estrogen (AE), is a selective estrogen receptor modulator that competes with the estradiol binding to the ER and is used to treat sex hormone-dependent breast cancer. Tam can be controlled by numerous variables, including growth factors. The ER/Tam complex, bound to the ER, brings in another protein known as a co-repressor, and this complex then binds to DNA and regulates gene expression. Researchers have also found that Tam acts as an agonist of E2 in some tissues, but as an antagonist in others. Tam has been studied in several fish, including Japanese medaka, tilapia, and zebrafish, with studies primarily on female virilization.
The main objective of modern aquaculture is to intentionally produce seedlings to preserve species, and create advanced fish species. Therefore, the research has focused on artificially inducing sexual maturation and ovulation. Studies have typically been conducted on methods such as; changing the concentrations of sex steroid hormones in rock fish (Sebastes inermis) by controlling the water temperature and photoperiod, the effects of water temperature and photoperiod on the development of Korean bullhead (Tachysurus fulvidraco) gonads, and inducing early sexual maturation in scallops by stimulating the water temperature. However, female fish generally exhibit problems with reduced immunity and slowed growth due to stress caused by the physiological changes required to produce eggs at the beginning of sexual maturation. Though studies have been reported on the induction and control of sexual maturity, no definite effects have been verified. Furthermore, though many experiments have been performed to determine the expression level of hormones by simply administering chemical factors into the fish body, no studies have reported sex control applicable to aquaculture farms.
The goal of this study was to secure and test technology for controlling (inhibiting) sexual maturation hormones such as maturation hormones through hormone regulation. To achieve this, first, we measured what is the difference Da, AI (Ana and Exe) and AE (Tam) affects inside a olive flounder (Paralichthys olivaceus), at several concentrations (in vitro). Second, we intraperitoneally injected Da, AI and AE into the bodies of female olive flounder (in vivo). This study confirmed the GnRH, Gth, VTG and ER mRNA expression in the hypothalamus, pituitary, liver and gonad due to the Da, AE and AI, as well as gonad weight index and histological changes.
Furthermore, it is necessary to compare these results and examine signaling pathways through chemical and genetic comparative analyses of the effects of Da, AI and AE treatment. This could be used as basic data for investigating the hormone secretion rate when injecting chemical substances into the body, and in terms of direct application to aquaculture farms, it is expected to be highly utilized in the industry.
Issued Date
Awarded Date
2021. 8
Alternative Author(s)
Kim, Ki Hyuk
제주대학교 대학원
대학원 해양생명과학과
Table Of Contents
제 1장. 종합 서론 1
제 2 장. 미성숙 암컷 넙치를 이용한 시상하부, 뇌하수체 및 간장 배양 세포에서 Dopamine, Anastrozole, Exemestane 및 Tamoxifen에 의한 성 성숙 관련 호르몬 물질의 억제 6
1. 서론 7
2. 재료 및 방법 9
2.1. 세포배양 9
2.2. 시료로부터의 RNA 추출 및 cDNA 합성 11
2.3. Quantitative qPCR 분석 12
2.4. 통계학적 분석 15
3. 결과 16
3.1. 시상하부, 뇌하수체 및 간장 배양세포 현미경 관찰 16
3.2. 시상하부 및 뇌하수체 세포에 Dopamine 투여 후 GnRH, Gth 및 ER mRNA level 변화 18
3.3. 간장 세포에 Aromatase inhibitors 투여 후 ER 및 VTG mRNA level 변화 23
3.3.1. Anastrozole에 의한 변화 23
3.3.2. Exemestane에 의한 변화 26
3.3.3. Tamoxifen에 의한 변화 29
4. 고찰 32
제 3 장. 미성숙 암컷 넙치를 이용한 Dopamine에 의한 성 성숙 억제 36
1. 서론 37
2. 재료 및 방법 39
2.1. 실험동물 및 Dopamine 처리 39
2.2. Dopamine 처리 후 샘플 40
2.3. 시상하부 및 뇌하수체에서의 Total RNA 추출 및 cDNA 합성 41
2.4. Quantitative PCR 분석 42
2.5. 생식소 중량 지수 관찰 42
2.6. 생식소 조직 관찰 43
2.7. 통계학적 분석 43
3. 결과 44
3.1. Dopamine 주사 후 시상하부 및 뇌하수체 GnRH, Gth 및 ER mRNA level 변화 44
3.2. Dopamine 주사 후 생식소 중량 지수(GSI) 변화 및 조직학적 변화 관찰 49
4. 고찰 52
제 4 장. 미성숙 암컷 넙치를 이용한 Aromatase inhibitors(AIs)에 의한 성 성숙 억제 54
1. 서론 55
2. 재료 및 방법 57
2.1. 실험동물 및 Aromatase inhibitors 처리 57
2.2. Aromatase inhibitors 처리 후 샘플 58
2.3. 간 및 생식소에서의 Total RNA 추출 및 cDNA 합성 59
2.4. Quantitative PCR 분석 60
2.5. 생식소 중량 지수 관찰 60
2.6. 생식소 조직 관찰 61
2.7. 통계학적 분석 61
3. 결과 62
3.1. Anastrozole 주사 후 ER 및 VTG mRNA level 변화 62
3.2. Anastrozole 주사 후 생식소 중량 지수(GSI) 변화 및 조직학적 변화 관찰 64
3.3. Exemestane 주사 후 ER 및 VTG mRNA level 변화 70
3.4. Exemestane 주사 후 생식소 중량 지수(GSI) 변화 및 조직학적 변화 관찰 72
4. 고찰 78
제 5 장. 미성숙 암컷 넙치를 이용한 Tamoxifen에 의한 성 성숙 억제 81
1. 서론 82
2. 재료 및 방법 84
2.1. 실험동물 및 Tamoxifen 처리 84
2.2. Tamoxifen 처리 후 샘플 85
2.3. 생식소 중량 지수 관찰 85
2.4. 간 및 생식소에서의 Total RNA 추출 및 cDNA 합성 86
2.5. Quantitative PCR 분석 87
2.6. 생식소 조직 관찰 88
2.7. 통계학적 분석 88
3. 결과 89
3.1. Tamoxifen 주사 후 ER 및 VTG mRNA level 변화 89
3.2. Tamoxifen 주사 후 생식소 중량 지수(GSI) 변화 및 조직학적 변화 관찰 91
4. 고찰 96
종합 고찰 99
참고 문헌 105
감사의 글 117
제주대학교 대학원
김기혁. (2021). 화학적 요법을 이용한 넙치의 성 성숙 억제 기술 개발에 관한 연구
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