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Determination of differential expression of sex-related genes in the brain-pituitary-gonad axis during the ovarian development of Japanese eel (Anguilla Japonica)

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Abstract
극동산 뱀장어(Aguilla japonica)는 일본, 대만, 중국, 필리핀, 배트남 그리고 한국을 포함한 동아시아에서 경제적적으로 중요한 부분을 차지하는 뱀장어과중 하나이다. 극동산 뱀장어는 강하회유성 생활사를 갖고 있으며, 대부분은 담수에서 생활하다가 산란을 위하여 하천에서 바다로 내려간다. 뱀장어는 양식분야에서 경제적으로 가치가 있는 주요어종이며, 자연산 실뱀장어를 활용한다. 뱀장어 양식을 위한 종묘는 모두 실뱀장어의 회유성을 활용하여 포획하여 양식한다. 지난 25년간, 자연산 실뱀장어의 남획으로 인하여 아시아와 유럽에서의 포획율이 급감하였다. 그러한 이유로 2014년, 세계자연보전연맹(the International Union for Conservation of Nature, IUCN)에서는 극동산 뱀장어를 멸종될 위기에 이른 종으로 분류를 하였다. 그러므로 자연산 실뱀장어의 증식을 위하여 인공적으로 번식을 유도하는 것이 최근 중요하게 여겨지고 있다.
이러한 이유로, 극동산 뱀장어의 생식과 이에 따른 기초적이고 근본적인 메커니즘을 이해하기 위하여 본 연구에서는 성선자극호르몬 (GnRH1, GnRH2), 성샘자극호르몬 (GTH)과 수용기 (FSHb, LHb, FSHr, LHr), 성호르몬과 수용기 (E2, ERa, ERb, ARa, ARb) 그리고 방향화효소 (CYP19A1, aromatase)를 뇌와 뇌하수체에서 발현을 분석하였다. 그리고 연어 뇌하수체 추출물 (SPE)를 극동산 뱀장어에 주입하여 생식선 조직으로부터 다양한 생식조직의 발단단계를 관찰하였다. 앞선 인자들을 분석하기 위하여 본 연구에서는 해부학적 관찰을 통해 perinucleolar stage (PN), nucleolar stage (N), early-vitellogenic stage (EV), mid-vitellogenic stage (MV), late-vitellogenic stage (LV), migratory nucleus stage (MN) 그리고 after ovulation (AO) stage로 총 7 단계로 성주기를 구별하였다. 뇌, 뇌하수체, 생식선 그리고 간 조직에서 RNA를 분리하여 대상 유전자의 발현을 측정하였다. 또한, 추가적으로 비텔로제닌 전사조절인자와 ERs를 간 조직에서 정량 PCR(qPCR)방법으로 측정하였다. 뇌조직으로부터 cDNA를 합성하였고, Kiss1 유전자의 부분 서열을 확보한 뒤에 Kiss1 과 Kiss1r mRNA 발현을 뇌와 생식선에서 qPCR 방법을 통하여 측정하였다.
본 연구에서 우리는 뱀장어과에서 최초로 Kiss1 유전자의 부분서열을 확인하였다. Kiss1 mRNA 발현량은 MN 단계에 유의적인 변화가 관찰되었으며, 뇌와 생식선에서는 Kiss1r의 발현이 두드러지게 관찰되었다.
뇌에서의 GnRH1과 GnRH2 mRNA는 다른 성주기에 비하여 MN 단계에서 높게 발현이 되었다. GnRH1의 발현과 유사하게 CYP19A1과 ERa mRNA 발현 또한 최종 성숙단계에서 유의적인 변화가 관찰되었다.
뇌하수체에서 FSHb 조절인자는 난황형성 시기와 MV 단계에 유의적으로 높게 나타났고 그 후 LV단계에서 AO단계까지 유의적으로 서서히 감소하는 것을 관찰되었다. 이와 대조적으로 LHb mRNA 발현은 휴지기에 비하여 LV, MN 그리고 AO 단계에서 유의적으로 상승되었고 MN 단계에서 가장 높게 관찰되었다.
생식선의 FSHr와 LHr 전사인자 수치는 난모세포가 발달됨에 따라 유사하게 나타나며 난모세포 성숙시기에는 두 전사인자 모두 유의적으로 높게 나타났다. 또한 CYP19A1 전사인자 수치는 다른 성숙 단계에 비하여 MN 단계에 급격히 유의적으로 상승하였다.
간에서는 vitellogenic을 주입하지 않은 암컷 뱀장어에서 상대적으로 매우 적은 수치의 Vtg 발현량을 확인하였다. 그러나 SPE를 주입 후 Vtg mRNA 발현은 상승하기 시작하여 LV 단계에서 가장 높게 나타났고 그 후 MN 단계에서 유의적으로 낮게 발현되었다. 이와 유사하게, ERs 전사인자 수치 또한 LV 단계에서 유의적으로 상승하였으나 MN 단계에서는 유의적인 감소가 관찰되지 않았다.
본 논문에서는 극동산 뱀장어에서 처음으로 생식선의 발달과 BPG 축과 관련하여 Kisspeptin (Kiss1) 체계에 대해서 관찰하였다. 추가적으로 우리는 극동산 뱀장어의 LH 주기를 결과적으로 완전하게 확인하였다. 또한, 다른 문헌에 근거하여 LV단계 이후에 aromatase가 감소할 것이라고 예상했지만 유의적인 변화를 관찰하지 못하였다.
Japanese eels (Anguilla Japonica) are one of the most commercially important eel groups, distributed in East Asian countries including Japan, Taiwan, China, Korea, Vietnam and Northern Philippines. This fish has a catadromous life cycle; which lives a part of the life cycle in the fresh water and migrates to sea for spawn. Eels are commercially produced by aquaculture of which majority is represented by of natural glass eels (Tsukamoto et al. 2009). Seedlings for eel aquaculture are totally dependent on capturing of glass eels during their upstream migration. During last 25 years, the rate of glass eel capturing has been reduced in Asia and Europe, because of the sharp decreasement of glass eel in natural habitats. Due to those reasons in 2014, Japanese eels were included in to the IUCN rest list as an endangered species. Therefore, the artificially induced breeding is essential to full fill the great demand of glass eel, and thereby develop the better method for eel production in order to protect the natural glass eels.
Hence, to understand the fundamental mechanisms involved in Japanese eel reproduction and provide a broad basis for comparative study of eel, we have analyzed the expression levels of gonadotropins (GnRH1, GnRH2), GTHs & their receptor (FSHb, LHb, FSHr, LHr), sex steroids & their receptor (E2, ERa, ERb, ARa, ARb) and aromatase (CYP19A1) enzyme in brain, pituitary and gonadal tissues during different gonadal development stages of immature and Salmon pituitary extract (SPE) injected Japanese eels. To analyze the transcriptional levels of aforementioned molecules, we have divided the complete estrus cycle into seven stages, including perinucleolar stage (PN), nucleolar stage (N), early-vitellogenic stage (EV), mid-vitellogenic stage (MV), late-vitellogenic stage (LV), migratory nucleus stage (MN) and after ovulation (AO) stage according to the histological observations. Brain, pituitary, gonad and liver tissues were isolated and RNA was extracted to investigate the above target gene expression levels. Furthermore, extra qPCRs were performed to examine the transcriptional modulations of vitellogenin (Vtg) and ERs in the liver tissue of above eels. Thereafter, cDNA was synthesised from brain tissues and amplified segment of Kiss1 gene partial sequence with degenerate primers using PCR, and examined the mRNA expression of Kiss1 in the brain and Kiss1r in brain & gonad during the oocyte development in Japanese eel by qPCR assays using gene specific primers.
To the best of our knowledge, we are the first group that cloned the partial sequence (120bp) of Kiss1 from the brain of anguillids. The Kiss1 mRNA expression was significantly elevated in the MN stage of the Japanese eel, where brain and gonad showed prominent Kiss1r expression.
The brain GnRH1 and GnRH2 mRNA were highly expressed during the MN stage compared to the other stages. Similar to the GnRH1 expression, CYP19A1 and ERa mRNA expression was also significantly pronounced in the brain during final oocyte maturation in Japanese eels.
In the pituitary, FSHb transcripts were abundant in vitellogenesis and significantly elevated in MV stage. After that, the expression was significantly and gradually decreased from LV to AO stages. In contrast to the FSH, pituitary LHb mRNA expression was significantly increased during LV, MN and AO stages than rest of the stages and reached the peak in MN stage. Pituitary CYP19A1 mRNA expression was also significantly elevated in LV and MN stages during gonadal development of Japanese eel.
The gonadal FSHr and LHr transcript level showed a synchronized fluctuation along the oocyte development in Japanese eel. Transcript levels of both genes were significantly increased during final oocyte maturation. Furthermore, CYP19A1 transcript levels were robustly and significantly increased in MN, compared to the other development stages in ovary of Japanese eels.
In the liver, comparatively very low Vtg expression was observed in un-injected (pre-vitellogenic) females. But after the SPE injection Vtg mRNA expression was started to increase and it was peaked in LV stage then significantly decreased in MN stage. As similar to the Vtg, ERs transcript levels also significantly increased in LV stage but not significantly decreased in MN stage.
Our present study provides the first insights into the kisspeptin (Kiss1) system of Japanese eel and its involvement in the BPG axis during gonadal development. In addition to that, our findings collectively complete the LH cycle of the Japanese eel. Furthermore, we speculate that failure to decrease the aromatase activity after the LV stage may be attributed to the suspension of the ovulation of Japanese eels.
Author(s)
Herath Mudiyanselage Viraj Udayantha
Issued Date
2015
Awarded Date
2016. 2
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000007417
Alternative Author(s)
헤라스 무디얀세라게 비라즈
Department
대학원 해양생명과학과
Advisor
이제희
Table Of Contents
1. Introduction 1
Role of the brain during oocyte maturation 8
Role of the pituitary during oocyte maturation 10
Role of the gonads during oocyte maturation 11
2. Material and Method 15
2.1. Animals 15
2.2. Induction of oocyte maturation and ovulation 15
2.3. Tissue collection 16
2.4. Histological observation 17
2.5. RNA extraction 20
2.6. Kiss1 partial sequence cloning 20
2.7. Quantification of Brain, Pituitary, Gonad and Liver target gene transcripts by real time PCR analysis. 21
2.8. ELISA analysis 24
2.9. Statistical analysis 24
3. Results 25
3.1. PART 1: The involvement of BPG-axis sex related genes into oocyte development in Japanese eel. 25
3.1.1. Involvement of the brain 25
3.1.2. Involvement of the Pituitary 30
3.1.3. Involvement of the Gonad 33
3.1.4. Involvement of the Liver 40
3.1.5 Involvement of serum 43
3.2. PART 2: Involvement of kisspeptin (Kiss1) and its receptor (Kiss1r) into BPG axis sex related genes during oocyte development in Japanese eel. 44
4. Discussion 47
4.1. Differential regulation of GnRH and GnRHr during oocyte development in Japanese eel. 47
4.2. Differential involvement of gonadotrophin (GTHs) subunit and their receptors during ovarian development 48
4.3. A single type CYP19A1 gene expression in BPG axis along gonadal development in the Japanese eel. 52
4.4. Differential contribution of steroid receptor subtypes during artificially induced ovarian maturation in Japanese eel 54
4.5. Liver dependent vitellogenesis during oocyte maturation. 56
4.6. Serum E2 involvement during oocyte development 57
4.7. Role of the Kisspeptine1 and Kiss1r during oocyte development in artificially matured Japanese eel 58
5. Conclusion 60
6. References 62
7. Acknowledgements 70
Degree
Master
Publisher
제주대학교 대학원
Citation
Herath Mudiyanselage Viraj Udayantha. (2015). Determination of differential expression of sex-related genes in the brain-pituitary-gonad axis during the ovarian development of Japanese eel (Anguilla Japonica)
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General Graduate School > Marine Life Sciences
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