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卵泡刺激素（FSH）和促黃體激素（LH）是脊椎動物體內的促性腺激素（GTH）。它們通過其相應的GTH受體（GTHR）－ FSH受體（FSHR）及LH/絨毛膜性腺激素受體（LHCGR），來調控雌性脊椎動物的主要性腺活動，如卵泡生成和類固醇生成。因此，GTHR的表達水平可控制卵泡細胞對於GTH的反應程度，從而影響脊椎動物的繁殖能力。然而，跟哺乳動物中的資料相比，這些受體的表達調控機制在硬骨魚類中仍然很模糊。此前，我們已經證明了斑馬魚卵泡之fshr和lhcgr的表達譜差異，顯示出lhcgr的表達滯後於fshr的表達。此表達時間之差異引申出兩條有趣的問題：一）甚麼激素能分別調節fshr和lhcgr的表達？ 二）這些調控的機制是甚麼？因此，我們發起本研究來解答這些問題。利用培養出來的斑馬魚卵泡細胞，我們展示了雌二醇（E2）是一個有力的GTHR調控激素。雖然E2同時刺激了fshr和lhcgr的表達，但E2對於lhcgr的表達調控效力遠遠比對fshr的高。由於雌激素核受體（nER）的特異拮抗劑（ICI 182,780）能完全抵消E2的效果，表明了E2是通過傳統的nER來直接促進了lhcgr的表達。有趣的是，不能穿越細胞膜的雌二醇-牛血清白蛋白偶聯複合物（E2-BSA）能完全模仿E2的效果，因此我們的證據提出這些nER可能位於細胞膜上。此外，我們運用各種藥劑發現了多種信號分子跟E2調控GTHR的能力有關，包括cAMP、PKA、PI3K、PKC、MEK、MAPK及p38 MAPK。當中以cAMP-PKA的信號傳導最有可能在E2的雙相調控效果起了直接作用，而E2的行動也極依賴其他信號分子的允許作用。除了E2，人絨毛膜促性腺激素（hCG; LH的類似物）、垂體腺苷酸環化酶激活多肽（PACAP）、表皮生長因子（EGF）和胰島素樣生長因子-I（IGF-I）也能有效地調節斑馬魚卵泡細胞的GTHR表達。hCG能大幅下調其受體lhcgr的表達，顯示hCG能令卵泡細胞對GTH脫敏。與此同時，PACAP能瞬時模仿hCG的行動，表明了PACAP很可能是hCG的瞬態下游信號。EGF是一個強烈抑制lhcgr表達的因子，而IGF-I是一個潛在的fshr表達增強因子，均說明了旁分泌因子對GTHR表達調控有關鍵作用。除了這些激素或因子的獨立調控作用，我們進一步發現了E2的效果可能會被它們覆蓋或調節。它們對nER的調控作用可能會造成這種現象。PACAP瞬時減少了esr2a及esr2b的表達量，而EGF則顯著地下調了esr2a。作為第一個在硬骨魚卵巢中對GTHR調控的全面研究，它無疑豐富了我們對卵泡生成過程中GTH的功能及GTHR表達調控的認識。此外，我們成功將目前的研究平台應用於雙酚A（BPA）的研究，進一步展示了本研究平台的潛力，有助於我們未來對各種內分泌干擾物（EDC）的作用機制進行研究。Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are the gonadotropins (GTHs), which bind to their cognate GTH receptors (GTHRs), FSH receptor (FSHR) and LH/choriogonadotropin receptor (LHCGR), to mediate major gonadal events in female vertebrates, including folliculogenesis and steroidogenesis. The expression level of GTHRs, therefore, controls the responsiveness of follicle cells to GTHs and hence governs the vertebrate reproduction.However, compared with the information in mammals, the expression control of these receptors in teleosts remains largely unknown. Previously, we have demonstrated the differential expression profiles of fshr and lhcgr in the zebrafish folliculogenesis, showing that lhcgr expression lags behind fshr expression. This temporal difference between fshr and lhcgr expression has raised two interesting questions: 1) What hormones regulate the differential expression of fshr and lhcgr? and 2) What are the control mechanisms of these regulations? The present study was initiated to answer these questions.With the primary zebrafish follicle cell cultures, we demonstrated that estradiol (E2) was a potent differential regulator of GTHRs. Although E2 increased both fshr and lhcgr expression, the up-regulatory potency of E2 on lhcgr was much greater than that on fshr. E2 directly promoted lhcgr expression via classical nuclear estrogen receptors (nERs) since nER-specific antagonist (ICI 182,780) completely abolished the E2 effect. Interestingly, our evidence suggested that these nERs could be localized on the plasma membrane because the membrane-impermeable form of estrogen (E2-BSA) fully mimicked the actions of E2. Furthermore, by applying various pharmaceutical agents, we revealed the involvement of multiple signaling molecules, including cAMP, PKA, PI3K, PKC, MEK, MAPK and p38 MAPK. The cAMP-PKA pathway likely played a direct role in the biphasic actions of E2 while the E2 actions were also greatly dependent on the permissive actions of other signaling molecules.Apart from the sex steroid E2, human chorionic gonadotropin (hCG; as a LH analogue), pituitary adenlyate cyclase-activating peptide (PACAP), epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) also significantly regulated GTHR expression in the zebrafish follicle cells. hCG drastically down-regulated its receptor, lhcgr, suggesting that hCG could desensitize the follicle cells to respond to GTH. Meanwhile, PACAP transiently mimicked the actions of hCG, indicating that PACAP was likely a transient downstream mediator of hCG. EGF was another strong suppressor of lhcgr expression while IGF-I was a potential fshr expression enhancer, which highlighted the crucial roles of paracrine factors in the regulation of GTHRs. In addition to the regulatory effect of these individual hormones or factors, we further revealed that the E2 action could be overridden or modulated by them. Their regulatory effects on the expression of nERs might contribute to this phenomenon. PACAP transiently reduced esr2a and esr2b expression while EGF significantly down-regulated esr2a.As the first comprehensive study of GTHR regulation in the teleost ovary, the present study certainly enriched our knowledge in the functions of GTHs and the expression control of GTHRs during folliculogenesis. By applying the current research platform on the study of bisphenol A (BPA), an endocrine-disrupting chemical (EDC), the present study further highlighted the potential of this research platform to contribute to the future action mechanism studies of various EDCs.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Liu, Ka Cheuk.Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.Includes bibliographical references (leaves 159-212).Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.Abstract also in Chinese.Abstract (in English) --- p.iAbstract (in Chinese) --- p.iiiAcknowledgement --- p.vTable of contents --- p.viList of figures and tables --- p.xiiSymbols and abbreviations --- p.xvChapter Chapter 1 --- General IntroductionChapter 1.1 --- Hypothalamic-pituitary-gonadal axisChapter 1.1.1 --- Overview --- p.1Chapter 1.1.2 --- Gonadotropin-releasing hormone --- p.1Chapter 1.2 --- FolliculogenesisChapter 1.2.1 --- Structure of ovarian follicles --- p.2Chapter 1.2.2 --- Stages of folliculogenesis --- p.3Chapter 1.3 --- Gonadotropins and gonadotropin receptorsChapter 1.3.1 --- History of teleost gonadotropin and gonadotropin receptors --- p.5Chapter 1.3.2 --- Structure --- p.6Chapter 1.3.3 --- Function --- p.7Chapter 1.3.4 --- GTH-GTHR specificity --- p.9Chapter 1.3.5 --- Signal transduction --- p.10Chapter 1.3.6 --- Expression profile of gonadotropin receptors --- p.11Chapter 1.3.7 --- Regulation of gonadotropin receptors --- p.12Chapter 1.4 --- Objectives and significances of the project --- p.14Chapter 1.5 --- Figure legends --- p.16Chapter 1.6 --- Figures --- p.18Chapter Chapter 2 --- Differential Regulation of Gonadotropin Receptors (fshr and lhcgr) by Estradiol in the Zebrafish Ovary Involves Nuclear Estrogen Receptors That Are Likely Located on the Plasma MembraneChapter 2.1 --- Introduction --- p.24Chapter 2.2 --- Materials and methodsChapter 2.2.1 --- Animals --- p.25Chapter 2.2.2 --- Hormones and chemicals --- p.26Chapter 2.2.3 --- Primary follicle cell culture and drug treatment --- p.26Chapter 2.2.4 --- Ovarian fragment incubation --- p.27Chapter 2.2.5 --- Total RNA extraction and real-time qPCR --- p.27Chapter 2.2.6 --- Western blot analysis --- p.27Chapter 2.2.7 --- SEAP reporter gene assay --- p.28Chapter 2.2.8 --- Data analysis --- p.28Chapter 2.3 --- ResultsChapter 2.3.1 --- Differential stimulation of fshr and lhcgr expression in ovarian fragments and follicle cells by estradiol but not testosterone --- p.28Chapter 2.3.2 --- Potentiation of follicle cell responsiveness to hCG by E2 pretreatment --- p.30Chapter 2.3.4 --- Evidence for transcription but not translation-dependent up-regulation of lhcgr by E2 --- p.30Chapter 2.3.5 --- Evidence for the involvement of nuclear estrogen receptors but not G protein-coupled estrogen receptor 1 (Gper) in E2-stimulated lhcgr expression --- p.31Chapter 2.3.6 --- Evidence for possible localization of estrogen receptors on the plasma membrane --- p.32Chapter 2.3.7 --- MAPK dependence of E2 effect on lhcgr expression --- p.32Chapter 2.4 --- Discussion --- p.33Chapter 2.5 --- Table --- p.38Chapter 2.6 --- Figure legends --- p.39Chapter 2.7 --- Figures --- p.43Chapter Chapter 3 --- Signal Transduction Mechanisms of the Biphasic Estrogen Actions in the Regulation of Gonadotropin Receptors (fshr and lhcgr) in the Zebrafish OvaryChapter 3.1 --- Introduction --- p.50Chapter 3.2 --- Materials and methodsChapter 3.2.1 --- Animals --- p.52Chapter 3.2.2 --- Hormones and chemicals --- p.52Chapter 3.2.3 --- Primary cell culture and drug treatment --- p.52Chapter 3.2.4 --- Total RNA extraction and real-time qPCR --- p.52Chapter 3.2.5 --- Fractionation of follicle cells --- p.52Chapter 3.2.6 --- Western blot analysis --- p.52Chapter 3.2.7 --- Statistical analysis --- p.53Chapter 3.3 --- ResultsChapter 3.3.1 --- Biphasic roles of cAMP-PKA pathway --- p.53Chapter 3.3.2 --- Effects of p38 MAPK inhibition --- p.54Chapter 3.3.3 --- Effects of PKC and PI3K inhibition --- p.54Chapter 3.4 --- Discussion --- p.55Chapter 3.5 --- Figure legends --- p.59Chapter 3.6 --- Figures --- p.61Chapter Chapter 4 --- Gonadotropin (hCG) and pituitary adenylate cyclase-activating peptide (PACAP) down-regulate basal and E2-stimulated gonadotropin receptors (fshr and lhcgr) in the zebrafish ovary via a cAMP-dependent but PKA-independent pathwayChapter 4.1 --- Introduction --- p.66Chapter 4.2 --- Materials and methodsChapter 4.2.1 --- Animals --- p.69Chapter 4.2.2 --- Hormones and chemicals --- p.69Chapter 4.2.3 --- Primary cell culture and drug treatment --- p.69Chapter 4.2.4 --- Total RNA extraction and real-time qPCR --- p.69Chapter 4.2.5 --- Statistical analysis --- p.69Chapter 4.3 --- ResultsChapter 4.3.1 --- Down-regulation of fshr and lhcgr by hCG --- p.69Chapter 4.3.2 --- Differential regulation of fshr and lhcgr by PACAP --- p.70Chapter 4.3.3 --- Inhibition of E2-regulated fshr and lhcgr expression by hCG --- p.71Chapter 4.3.4 --- Suppressive effects of PACAP on E2-induced fshr and lhcgr expression --- p.71Chapter 4.3.5 --- Role of cAMP in hCG and PACAP actions --- p.72Chapter 4.4 --- Discussion --- p.73Chapter 4.5 --- Figure legends --- p.78Chapter 4.6 --- Figures --- p.80Chapter Chapter 5 --- Paracrine regulation of gonadotropin receptors (fshr and lhcgr) by ovarian growth factors: epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I)Chapter 5.1 --- Introduction --- p.85Chapter 5.2 --- Materials and methodsChapter 5.2.1 --- Animals --- p.88Chapter 5.2.2 --- Hormones and chemicals --- p.88Chapter 5.2.3 --- Primary cell culture and drug treatment --- p.88Chapter 5.2.4 --- Total RNA extraction and real-time qPCR --- p.88Chapter 5.2.5 --- Statistical analysis --- p.88Chapter 5.3 --- ResultsChapter 5.3.1 --- Biphasic down-regulation of lhcgr by EGF --- p.89Chapter 5.3.2 --- Evidence for EGFR involvement --- p.89Chapter 5.3.3 --- Minor role of MEK-MAPK3/1 pathway in the EGF effect on lhcgr expression --- p.90Chapter 5.3.4 --- Up-regulation of fshr by IGF-I --- p.90Chapter 5.3.5 --- Evidence for IGF-IR involvement --- p.91Chapter 5.3.6 --- Role of PI3K-Akt pathway in IGF-I action --- p.91Chapter 5.3.7 --- Role of EGF and EGFR in E2-induced GTHR expression --- p.91Chapter 5.3.8 --- Role of IGF-I and IGF-IR in E2-induced GTHR expression --- p.91Chapter 5.4 --- Discussion --- p.92Chapter 5.5 --- Figure legends --- p.98Chapter 5.6 --- Figures --- p.100Chapter Chapter 6 --- Regulation of estrogen receptor subtypes (esr1, esr2a and esr2b): a possible mechanism to modulate estradiol-stimulated lhcgr expression in the zebrafish ovaryChapter 6.1 --- Introduction --- p.107Chapter 6.2 --- Materials and methodsChapter 6.2.1 --- Animals --- p.110Chapter 6.2.2 --- Hormones and chemicals --- p.110Chapter 6.2.3 --- Staging ovarian follicles --- p.110Chapter 6.2.4 --- Primary cell culture and drug treatment --- p.110Chapter 6.2.5 --- Total RNA extraction and real-time qPCR --- p.110Chapter 6.2.6 --- Statistical analysis --- p.111Chapter 6.3 --- ResultsChapter 6.3.1 --- Expression profiles of estrogen receptors (ERs) in zebrafish folliculogenesis --- p.111Chapter 6.3.2 --- Homologous regulation of nERs by E2 --- p.111Chapter 6.3.3 --- Regulation of nERs by endocrine hormones (hCG and PACAP) --- p.112Chapter 6.3.4 --- Regulation of nERs by ovarian paracrine growth factors (EGF and IGF-I) --- p.112Chapter 6.3.5 --- Role of cAMP in nER regulation --- p.113Chapter 6.3.6 --- Role of PKA in nER regulation --- p.113Chapter 6.4 --- Discussion --- p.114Chapter 6.5 --- Figure legends --- p.119Chapter 6.6 --- Figures --- p.121Chapter Chapter 7 --- Estrogenic Action Mechanisms of Bisphenol AChapter 7.1 --- Introduction --- p.127Chapter 7.2 --- Materials and methodsChapter 7.2.1 --- Animals --- p.129Chapter 7.2.2 --- Hormones and chemicals --- p.129Chapter 7.2.3 --- Primary cell culture and drug treatment --- p.129Chapter 7.2.4 --- Total RNA extraction and real-time qPCR --- p.129Chapter 7.2.5 --- Statistical analysis --- p.130Chapter 7.3 --- ResultsChapter 7.3.1 --- Expression of fshr and lhcgr interfered by BPA --- p.130Chapter 7.3.2 --- Signaling mechanism of BPA-induced lhcgr up-regulation --- p.130Chapter 7.3.3 --- Dependence of transcription and translation in BPA-induced lhcgr expression --- p.131Chapter 7.3.4 --- Evidence for the involvement of nuclear estrogen receptors in the BPA actions --- p.131Chapter 7.3.5 --- Interference on E2-induced lhcgr expression by BPA --- p.131Chapter 7.4 --- Discussion --- p.132Chapter 7.5 --- Figure legends --- p.136Chapter 7.6 --- Figures --- p.138Chapter Chapter 8: --- General DiscussionChapter 8.1 --- Estradiol as a differential regulator of gonadotropin receptors --- p.143Chapter 8.2 --- Conserved role of estradiol with differential action mechanisms in lhcgr regulation of mammals and teleosts --- p.144Chapter 8.3 --- Involvement of classical estrogen receptors that are likely located on the plasma membrane --- p.145Chapter 8.4 --- Biphasic response of lhcgr to estradiol and the underlying signal transduction mechanisms --- p.145Chapter 8.5 --- Desensitization of follicle cells to gonadotropins by hCG --- p.146Chapter 8.6 --- Paracrine control of gonadotropin receptors by ovarian growth factors --- p.147Chapter 8.7 --- Interaction of the estrogen action with other endocrine and paracrine signals --- p.148Chapter 8.8 --- Action mechanism studies of an endocrine-disrupting chemical: bisphenol A --- p.150Chapter 8.9 --- Conclusion --- p.151Chapter 8.10 --- Figure legends --- p.153Chapter 8.11 --- Figures --- p.155References --- p.15