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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/34563

Title: 點帶石斑魚(Epinephelus coioides)早期腦部發育中神經性類固醇的合成與雌激素訊息之探討
Neurosteroids synthesis and estrogen signaling in the early brain of orange-spotted grouper, Epinephelus coioides
Authors: Ganesan Nagarajan
羅睿哲
Contributors: NTOU:Department of Aquaculture
國立臺灣海洋大學:水產養殖學系
Keywords: 石斑魚;大腦發育;類固醇合成酶;雌激素;性腺分化
Grouper;Brain development;Steroidogenic enzymes;Estrogen;Gonadal sex differentiation;Cyp19a1b;SF-1;Dax-1
Date: 2013
Issue Date: 2013-10-07T02:42:37Z
Abstract: 真骨魚 (telesot fish)的腦部有著其獨特的神經內分泌調控機制;其神經生成的過程與扮演著神經前驅細胞的放射性神經膠細胞連結著,而芳香酶的濃度也比哺乳動物高出一百到一千倍。目前已確定其腦部可以產生類固醇,並且在發育的過程中扮演著關鍵的角色。在真骨魚性腺分化過程中,腦部發育的早期受到許多神經內分泌因子及受體的調控,而這些調控的機制還有許多未被了解的地方。在這篇論文中,我們以點帶石斑魚 (Epinephelus coioides)為動物模型來研究上述的機制。點帶石斑魚是一種雌性先熟雌雄同体的魚類;在生命週期的前六年為雌性生殖而在約第七年的時候開始轉換為雄性生殖 我們假設初期點帶石斑魚的腦部可以產生一定量的神經性雌二醇,因而調控下游訊息傳遞以及神經性類固醇的表現,進而影響性腺分化。我們首先選殖及分析star (steroidogenic acute regulatory protein,類固醇生成急性調控蛋白) 、cyp11a1 (cytochrome P450 side chain cleavage,細胞色素P450支鏈裂解酵素)、hsd3b1 (3β-hydroxysteroid dehydrogenase,3β-羥類固醇去氫酶)、cyp17a1 (cytochrome P450c17,細胞色素P450c17)、arcyp19a1b (cytochrome P450 aromatase,细胞色素P450芳香化酶)這些在顳腦中與神經性雌激素生合成有關的主要基因的表現,同時也利用即時定量聚合酶連鎖反應觀察不同發育年齡(孵化後九十至一百八十天)時前腦、中腦、下視丘及後腦中雌激素受體 (α、β1、β2)、雌激素細胞膜受體 (GPR30)以及雄激素受體。結果指出除了上述除了雄激素受體以外的基因在性腺分化時均有顯著表現上升的情形. 而細胞增生活性指標pcna (proliferating cell nuclear antigen, 細胞增生核抗原)的表現在孵化後一百一十至一百五十天的也觀察到表現量上升。 另外,我們也發現相較於其他的時間點,在孵化後一百一十及一百二十天時前腦內的有著較高濃度的芳香酶以及雌二醇,更進一步的,我們觀察到在前腦及下視丘中、雌二醇可提高cyp17a1及cyp19a1b的表現,而在中腦中則是可提高hsd3b1的表現,至於cyp11a1則是在所有觀察的腦區中均無變化。而在中腦腦室、前視區及間腦腦室內利用外源表現的雌二醇也同樣的可以提高Cyp19a1b的表現,證明了雌二醇確實可調控類固醇激素基因。原位雜交的分析結果則顯示不管是在前腦、下視丘或是後腦,cyp11a1、hsd3b1及cyp17a1 均擁有相似的表現量,而在小腦中,hsd3b1則有著較高的表現,在放射性神經膠細胞中則發現cyp19a1b的基因與蛋白質量表現均有提高。綜合以上的結果,我們認為cyp19a1b只存在放射性神經膠細胞中。Cyp19a1b與PCNA的免疫螢光組織染色結果則顯示絕大部分增生的細胞均為放射性神經膠細胞。最後,結合了Cyp19a1b免疫螢光染色以及cyp11a1、hsd3b1和cyp17a1螢光原位雜合的結果則顯示cyp11a1, hsd3b1 and cyp17a1的mRNA存在於有著Cyp19a1b活性的放射性神經膠細胞中,代表cyp11a1, hsd3b1 and cyp17a1 mRNA來自於放射性神經膠細胞。 我們同時也分析了早期腦部中sf-1以及 dax-1這兩種孤兒核受體(orphan nuclear receptors)的表現與孵化後不同天數至性腺分化時促類固醇生成酶(star、cyp11a1、hsd3b1、cyp17a1與cyp19a1b)以及雌激素受體(ERα、ERβ1、與ERβ2)基因表現程度的關聯性。定量聚合酶連鎖反應顯示sf-1 mRNA與上述性腺分化促類固醇生成酶有顯著的正相關,而dax-1則剛好相反,代表這些孤兒核受體確實在腦中確實有其功能性。而在不同的發育時期時,sf-1以及 dax-1會在下視丘中表現,另外,在下視丘腹內側細胞中利用原位雜交發現star、 sf-1 與dax-1的大量表現,而在活體中作用雌二醇之後,雖然並未發現dax-1 的表現有所變化,但star 與 sf-1 的表現量確實有明顯的上升。最後,實驗數據也表示在早期腦部發育時,精催產素(arginine vasotocin) 的產生神經內分泌功能可能是透過神經類固醇與雌激素的訊息傳遞來執行。綜合以上,我們的結果證明了點帶石斑魚腦中擁有著所有類固醇生成基因、類固醇生成因子、固醇類性荷爾蒙受體、其他固醇以及芳香酶的活性。更進一步的,我們的結果證明了上述分子的基因從孵化後腦部發育早期就開始活化並轉錄,並在整個需要依賴性荷爾蒙的腦部發育過程中有著重要的功能,同時,研究結果也證明了點帶石斑魚的腦中有著產生所需要雌二醇的機制,因此擁有著在性腺分化時調控腦部發育的能力,而也正因為如此,點帶石斑魚的腦部發育的高峰期恰巧位於性腺發育的時候。
Since the brain of telesot fish exhibits a significant degree of neuroendocrine regulation, notably an intense neurogenic process linked to the persistence of radial glial cells acting as neural progenitors and remarkably high activity of brain aromatase, which is 100-1000 times higher than mammals, it has become clear that the brain is a true steroidogenic organ and important key player during development. In teleost, the neuroendocrine regulation of early brain development during gonadal sex differentiation is a complex phenomena regulated by various neuroendocrine factors and receptors during a critical period of development, which remains largely unknown. In the present study, we selected orange-spotted grouper Epinephelus coioides as a primary animal model to investigate the molecular mechanism underlying early brain development during gonadal sex differentiation. Groupers are protogynous hermaphrodite and functional females for the first six years of life and start to sex change around the age of seven years. In the present study, we hypothesized that the grouper brain has the capacity to produce a significant amount of neural estrogen (E2) in the early brain, and this locally produced E2 could auto-regulate the functional peak of neurosteroidogenesis and estrogen signaling in the brain, which may have an association with the occurrence of gonadal sex differentiation in this species. To test this hypothesis, we first molecular cloned and analyzed the temporal expression patterns of key genes involved in neural estrogen biosynthesis pathway includes star (steroidogenic acute regulatory protein), cyp11a1 (cytochrome P450 side chain cleavage), hsd3b1 (3β-hydroxysteroid dehydrogenase), cyp17a1 (cytochrome P450c17) and cyp19a1b (cytochrome P450 aromatase), and sex steroid nuclear receptors for estrogen (ERα, ERβ1 and ERβ2), androgen (AR) and the plasma membrane-associated estrogen receptor (GPR30) were also studied at different developmental ages (from 90 to 180-dah: days after hatching) in different brain regions (forebrain, midbrain, hypothalamus and hindbrain) using quantitative real-time PCR (q-PCR) and RT-PCR analyses. The results indicated that the mRNA expressions of all key genes and estrogen receptors except for AR were significantly increased in the brain during the period of gonadal sex differentiation. Moreover, pcna transcripts (a marker for cell proliferation activity) were higher in the early brain at 110–150 dah. Brain aromatase activity and E2 concentration levels, but not testosterone (T), were higher in the forebrain at 110-dah and 120-dah, respectively compared to other ages. Further, exogenous E2 upregulated cyp17a1 and cyp19a1b transcripts in the forebrain and hypothalamus, however, E2 did not alter cyp11a1 and hsd3b1 transcripts in any brain regions except hsd3b1 expression in the midbrain studied. Exogenous E2 also upregulated Cyp19a1b immunoreactivity in the telencephalic ventricle, preoptic area and diencephalic ventricle compared to their control brain, suggesting E2 regulation on steroidogenic genes. Furthermore, in situ hybridization (ISH) analysis revealed that cyp11a1, hsd3b1 and cyp17a1 transcripts were localized from the forebrain, hypothalamus and hindbrain with an overall similar expression pattern; at the same time, hsd3b1 transcript was higher in the cerebellum compared to other genes. High density in the cyp19a1b/Cyp19a1b expression was detected in radial glial cells. This is possibly concluded that the expression of grouper cyp19a1b/Cyp19a1b is restricted to radial glial cells. Besides, dual-labeled fluorescence immunohistochemistry (FIHC) of Cyp19a1b and PCNA further show that most of the proliferative cells were corresponded to radial glial cells. Moreover, by combining Cyp19a1b FIHC with florescence ISH (FISH) of cyp11a1, hsd3b1 and cyp17a1, we showed that sub-cellular localization of cyp11a1, hsd3b1 and cyp17a1 transcripts, in partial if not all, appeared to be in Cyp19a1b immunoreactive radial glial cell soma or process, suggesting mRNA export through radial glial cells. Next, orphan nuclear receptors such as sf-1 and dax -1 expressions were studied in the early brain in relation to a wider suite of neurosteroidogenic enzyme genes (star, cyp11a1, hsd3b1, cyp17a1 and cyp19a1b) and estrogen receptors (ERα, ERβ1 and ERβ2) during different post hatching developmental ages and analyzed them against the timing of gonadal sex differentiation. q-PCR results showed that sf-1 mRNA expression was significantly increased together with other key steroidogenic genes during gonadal sex differentiation whereas dax-1 expression was decreased as opposite to sf-1, indicating functional interaction of these orphan nuclear receptors in the grouper brain. sf-1 and dax-1 transcripts were localized in the hypothalamus during different developmental ages by in situ hybridization analysis. Localization of star, sf-1 and dax-1 were highly detected in the cells of the ventromedial hypothalamus (VMH) and in vivo E2 treatment was significantly upregulated only star and sf-1 expressions in the VMH. However, there was no difference detected in the dax-1 expression. Furthermore, the present study also indicates that neurosteroids and estrogen signaling may mediate some of the neuroendocrine functions of AVT during the early brain development. Collectively, the present study demonstrated that the early grouper brain exhibits all necessary changes in the expression of steroidogenic genes, factors, sex steroid receptors, brain steroid concentration and brain aromatse activity. These data provide the evidence that these genes are functional during early post-hatching development in the brain, and have a distinct contribution to different aspects of sex hormone-dependent brain development. Overall, this study demonstrated that the orange-spotted grouper brain possesses all required mechanisms for the local production of E2 which possibly regulates early brain development during gonadal sex differentiation. Therefore, a peak time of development in the early brain is suggested to occur during gonadal sex differentiation in the grouper.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0D94330008
http://ntour.ntou.edu.tw/handle/987654321/34563
Appears in Collections:[水產養殖學系] 博碩士論文

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