雙酚-A(bisphenol-A, BPA)廣泛地應用於人類的日常生活中，它是具有雌激素活性的化學物質。近年來，它對人類健康的影響引起了廣泛的關注。研究表明，胎儿期或嬰儿期暴露於BPA中會造成女性卵巢發育紊亂、青春期提前和性早熟。除青鱂、花溪鱂、大菱鮃、金魚、鯉、褐鱒魚和斑馬魚外，BPA對其它硬骨魚生殖功能的影響鮮見報導。而BPA對硬骨魚生殖軸的影響方面更是知之甚少。此外，在斑馬魚中，生殖發育的主要事件，包括性腺分化和青春期開始時間，以及青春期腦垂體激素如GTHs（FSH和LH）的時空表達模式仍然是一個未知數。因此，弄清這些問題不僅有助於理解GTHs在早期性發育中的作用，也有利於研究BPA對斑馬魚生殖軸的影響。 / 利用組織學分析方法、雙色熒光原位雜交（FISH）技術、實時定量PCR技術、蛋白質組分析和在體（活體）試驗，本文研究了斑馬魚性別分化和青春期開始的時間、腦垂體激素FSH（fshb）和LH（lhb）亞基在個體發育過程中特別是性別分化和青春期開始階段的表達模式；同時探討了在青春期之前BPA對斑馬魚生殖軸的影響。從早期的發育階段至性成熟的各個時間點分別收集樣本，用以建立斑馬魚性別分化和青春期開始的時間表及GTHs在其個體發育過程中的表達譜。利用組織學方法檢測性腺發育階段。斑馬魚的頭部（含整個腦和腦垂體）則用於FISH分析以了解其GTHs的表達譜。為分析BPA對斑馬魚生殖功能的影響，將受精後20天的幼魚暴露於濃度為10 μM的BPA中，同時以17β-雌二醇（E2, 10 nM）和睾酮（T, 10 nM） 作為陽性對照。處理20天后分別取其腦、腦垂體、肝臟和卵巢進行組織學、原位雜交、基因表達分析和蛋白質組學分析。 / 雌性斑馬魚青春期的第一個形態學標誌是從初級生長（PG）卵泡第一次轉變為/過渡到卵黃發生階段（PV）；我們的結果表明此過程大約發生在受精後第45天。同時，青春期的啟動似乎高度依賴於身體的生長。另外，原位雜交結果顯示，fshb基因的表達遠早於lhb基因, 在受精後4天就能檢測到fshb的mRNA信號（~2-3細胞/腦垂體）；而lhb的表達則在性別分化時約受精後25天才可檢測到。有趣的是， 表達lhb的細胞數量在青春期前非常少（~5-6 細胞/腦垂體），而青春期期間及之後則大幅增加。相反，在青春期之前，大量細胞表達fshb；青春期期間，表達fshb的細胞數量僅略有增加。因此，我們的結果顯示LH在雌性斑馬魚青春期啟動中具有重要的作用。另一方面，雖然BPA和E2可促進斑馬魚卵巢的分化，但它們亦能顯著抑制卵巢的生長發育。同時，BPA和E2都能明顯抑制垂體fshb的表達，這與其對卵巢大小的抑製作用似乎有密切的相關性。T對垂體fshb的表達無明顯的影響。進一步的結果顯示，BPA和E2不會影響GTHs上游調節基因（包括kiss1, kiss2, gnrh2 和gnrh3）的表達。在肝臟中，BPA和E2顯示出不同的效應。E2能誘導斑馬魚肝增生而導致其腹部水腫；而BPA暴露處理則無此效應。這些結果表明BPA具有雌激素的作用，可影響雌性斑馬魚的生殖功能，但從本研究所使用的劑量效應來看，它卻並不完全具有E2的全部效應。 / Being an estrogenic chemical and its ubiquitous presence in our daily lives, the effects of bisphenal A (BPA) on human health have received tremendous attention in recent years. Studies on the effects of BPA on female reproductive system have shown that early exposure to BPA during the prenatal or postnatal period impairs reproductive functions, including disruption of ovarian development, advanced pubertal onset, and the induction of an early, and persistent estrus. In teleost fish, few studies have been reported on the effects of BPA on reproductive function, except in medaka, Kryptolebias marmoratus, turbot, goldfish, common carp, brown trout and zebrafish. Despite these studies, the effects of BPA on reproductive axis remain largely unknown in teleost fish. On the other hand, in the zebrafish model, the major developmental events of reproduction, including the timing of puberty onset, the spatiotemporal expression patterns of key pituitary hormones such as GTHs (FSH and LH) during gonadal differentiation and puberty development remain largely unknown. Therefore, the information on these issues in zebrafish not only is valuable for understanding the roles of GTHs in early sexual development; also facilitate our study on the effects of BPA on the reproductive axis in the zebrafish. / Using histology analysis, double-colored fluorescent in situ hybridization (FISH), real-time qPCR, proteomic analysis and in vivo treatment, this study was undertaken to explore the timing of sex differentiation and puberty onset, the ontogenic expression patterns of FSH (fshb) and LH (lhb) subunits in the zebrafish pituitary with particular emphasis on the stage of sexual differentiation and puberty onset, and the effects of BPA on the reproductive axis in zebrafish during prepubertal period. To define the timeline of sex differentiation and puberty onset, and the ontogenic expression profiles of GTHs, the zebrafish were collected at different time points from early development stage to sexual maturation. The gonadal developmental stage was analyzed by histological examination. For the expression profiles of GTHs, the head of each fish including the brain and pituitary was sampled for FISH analysis. To investigate the influence of BPA on the reproductive function, Juvenile zebrafish of 20 day post-fertilization (dpf) were exposed to BPA (10 μM) for 20 days followed by sampling the brain, pituitary, liver and ovary for histological, in situ hybridization, expression analyses and proteomic analyses at 40 dpf. 17β-estradiol (E2, 10 nM) and testosterone (T, 10 nM) were also used as a positive control. / In female zebrafish, the first morphological sign for puberty is the first wave of follicle transition from the primary growth (PG) to previtellogenic stage (PV), our results showed that it occurs around 45 day post fertilization (dpf). Meanwhile, the puberty onset was highly depending on the somatic growth. The expression of fshb was much earlier than that of lhb with its mRNA signal detectable (2-3 cells/pituitary) shortly after hatching (4 dpf). In contrast, lhb expression became detectable at the time of sex differentiation (~25 dpf). Interestingly, the number of lhb-expressing cells was very low (~5-6 cells/pituitary) before puberty but increased dramatically during and after puberty onset. In contrast, the expression of fshb was abundant before puberty with only a slight increase in cell number during puberty onset. Our result strongly suggests an important role for LH at the puberty onset of female zebrafish. On the other hand, although BPA and E2 both promoted ovarian differentiation, they significantly suppressed the ovarian growth afterwards in the zebrafish. Meanwhile, both BPA and E2, but not T, dramatically decreased the expression of fshb in the pituitary, which was well correlated with the suppression of ovarian size. However, the expression of the upstream regulators of GTHs, including kiss1, kiss2, gnrh2 and gnrh3, was not affected by BPA and E2. Interestingly, at the liver level, BPA and E2 displayed different effects. E2 induced abdominal swelling due to a significant hepatic hyperplasia. However, BPA exposure had no such effect on the liver. These results indicate that BPA has estrogenic effects on female reproduction, but it does not mimic E2 in all aspects, at least for the dose tested in the present study. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Weiting. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 93-120). / 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.I / Abstract in Chinese --- p.III / Acknowledgement --- p.V / Table of contents --- p.VI / List of figures and tables --- p.IX / Symbols and abbreviation --- p.XI / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Pituitary --- p.1 / Chapter 1.1.1 --- Structure --- p.1 / Chapter 1.1.2 --- Functions in reproduction --- p.2 / Chapter 1.2 --- Gonadotropins --- p.2 / Chapter 1.2.1 --- Structure --- p.2 / Chapter 1.2.2 --- Expression profile --- p.2 / Chapter 1.2.3 --- Regulation --- p.4 / Chapter 1.3 --- Gonadal development --- p.8 / Chapter 1.3.1 --- Sex differentiation --- p.8 / Chapter 1.3.2 --- Puberty initiation --- p.10 / Chapter 1.4 --- Objectives of the present study --- p.12 / Chapter Chapter 2 --- Puberty Initiation is Dependent on the Body Growth but not Age in Female Zebrafish / Chapter 2.1 --- Introduction --- p.15 / Chapter 2.2 --- Materials and methods --- p.16 / Chapter 2.2.1 --- Animals --- p.16 / Chapter 2.2.2 --- Sampling and measurement of body weight and body length --- p.16 / Chapter 2.2.3 --- Paraffin section and H & E staining --- p.17 / Chapter 2.2.4 --- Statistical analysis --- p.17 / Chapter 2.3 --- Results --- p.17 / Chapter 2.3.1 --- Growth curve of zebrafish during gonadal differentiation and maturation --- p.17 / Chapter 2.3.2 --- Gonadal differentiation in the zebrafish --- p.17 / Chapter 2.3.3 --- Puberty onset in female zebrafish --- p.18 / Chapter 2.3.4 --- Relationship of body growth and puberty initiation in female zebrafish --- p.19 / Chapter 2.4 --- Discussion --- p.19 / Chapter Chapter 3 --- Ontogenic Expression Profiles of Gonadotropins (fshb and lhb) and Growth Hormone (gh) During Sexual Differentiation and Puberty Onset in Female Zebrafish / Chapter 3.1 --- Introduction --- p.29 / Chapter 3.2 --- Materials and methods --- p.30 / Chapter 3.2.1 --- Animals --- p.30 / Chapter 3.2.2 --- Sampling --- p.31 / Chapter 3.2.3 --- Histological examination --- p.31 / Chapter 3.2.4 --- Total RNA isolation and reverse transcription --- p.32 / Chapter 3.2.5 --- Fluorescent double-colored in situ hybridization --- p.32 / Chapter 3.2.6 --- Microinjection of morpholino knockdown --- p.32 / Chapter 3.2.7 --- Real-time qPCR quantification of fshb, lhb and gh expression --- p.33 / Chapter 3.2.8 --- Data analysis --- p.34 / Chapter 3.3 --- Results --- p.34 / Chapter 3.3.1 --- Detection of fshb, lhb and gh expression in the pituitary of adult zebrafish --- p.34 / Chapter 3.3.2 --- Expression of fshb, lhb and gh before gonadal differentiation (4-25 dpf) --- p.34 / Chapter 3.3.3 --- Expression of fshb, lhb and gh during gonadal differentiation (22-25 dpf) --- p.35 / Chapter 3.3.4 --- Expression of fshb, lhb and gh during puberty period (~45 dpf) --- p.35 / Chapter 3.3.5 --- Phenotypes of fshb-MO and lhb-MO zebrafish in early development36 --- p.36 / Chapter 3.4 --- Discussion --- p.37 / Chapter Chapter 4 --- Neonatal Exposure to 17β-Estradiol or Bisphenol A Promotes Ovarian Differentiation but Suppresses Its Growth Probably via Inhibiting Follicle-Stimulating Hormone Expression / Chapter 4.1 --- Introduction --- p.55 / Chapter 4.2 --- Materials and methods --- p.57 / Chapter 4.2.1 --- Animals --- p.57 / Chapter 4.2.2 --- In vivo treatment and hormone replacement --- p.57 / Chapter 4.2.3 --- Sampling --- p.58 / Chapter 4.2.4 --- Total RNA isolation and reverse transcription --- p.58 / Chapter 4.2.5 --- Fluorescent double-colored in situ hybridization --- p.58 / Chapter 4.2.6 --- Real-time qPCR quantification --- p.59 / Chapter 4.2.7 --- Protein extraction and quantification --- p.59 / Chapter 4.2.8 --- Two-dimensional electrophoresis --- p.60 / Chapter 4.2.9 --- Staining --- p.61 / Chapter 4.2.10 --- In-gel digestion --- p.61 / Chapter 4.2.11 --- Mass spectrometry --- p.61 / Chapter 4.2.12 --- Data analysis --- p.61 / Chapter 4.3 --- Results --- p.62 / Chapter 4.3.1 --- E2 had distinct effect on the body growth and behavior --- p.62 / Chapter 4.3.2 --- BPA and E2 increased female ratio but suppressed ovarian growth in the zebrafish --- p.62 / Chapter 4.3.3 --- BPA and E2 shut down fshb but increased lhb expression in the pituitary without altering the expression of GnRH and kisspeptin in the hypothalamus --- p.63 / Chapter 4.3.4 --- E2 but not BPA induced hepatic hyperplasia --- p.64 / Chapter 4.4 --- Discussion --- p.65 / Chapter Chapter 5 --- General Discussion

生長和繁殖這兩個最主要的生理過程在脊椎動物中是密切相關的。生長主要是由腦-垂體-肝軸，即促生長激素釋放激素/垂體腺苷酸環化酶激活肽（GHRH/ PACAP-生長激素（GH）-胰島素樣生長因子（IGFs）軸所控制。值得一提的是，所有與這個神經內分泌軸相關的基因在卵巢中都有表達。這表明一個有功能的微型軸很可能存在於卵巢中。我們著重研究PACAP，該軸的上游因子，來揭示卵巢中這個微型軸的存在和功能。 / PACAP是一種最初從羊的下丘腦純化出來的夠能刺激cAMP分泌的神經肽，研究表明它也存在於如卵巢在內的其它各種外圍組織中。在斑馬魚中，兩種形式的PACAP（PACAP38-1，adcyap1a; PACAP38-2，adcyap1b）和三個 PACAP受體（PAC1-R，adcyap1r1; VPAC1-R，vipr1和VPAC2-R，vipr2）均在卵巢中表達。為了確定PACAP系統在斑馬魚的卵巢中有重要作用，我們首先對 PACAP的配體和三個受體在濾泡中的空間分佈進行了研究。此外，為了研究PACAP系統的潛在作用，我們還分析了PACAP的配體和受體在濾泡發育和成熟時期的表達情況。PACAP系統在濾泡細胞中時空表達的數據顯示，PACAP可能在調節濾泡發育和成熟中發揮雙重作用，這雙重作用是通過PACAP作用於不同的受體上完成的。卵母細胞體外成熟實驗的結果顯示PACAP可以促進完整的濾泡卵母細胞的成熟，但抑制裸露的卵母細胞體外自發成熟，這也進一步支持了我們的假說。 / 我們以前的研究表明，在原代培養的斑馬魚濾泡細胞中，垂體促性腺激素（HCG）可以顯著提高PACAP（PACAP38-2）的表達。因此，PACAP很可能是垂體促性腺激素控制卵巢功能的下游調節者。我們從幾個方面來驗證我們的假設。首先，由於激活素/結合蛋白系統是公認的垂體促性腺激素（HCG）的下游調節者，我們研究了PACAP對該系統表達的調控。研究結果表明，PACAP不僅能模仿促性腺激素對激活素/結合蛋白系統表達的調控，同時也刺激激活素介導的卵母細胞的成熟。PACAP和hCG選擇同樣的信號通路對激活素/結合蛋白系統進行調控進一步證實 PACAP的下游調節作用。其次，卵巢內源性生長因子，表皮生長因子EGF對PACAP調控激活素/結合蛋白系統表達的影響和其對hCG調控該系統表達的影響是一樣的。表皮生長因子可以作用於其膜上的受體並且使用MEK途徑來調節PACAP對激活素/結合蛋白系統的表達的調控。第三，我們研究了PACAP對激素生成的影響。芳香化酶是激素生成中一個十分重要的酶，它可以將睾酮轉化成雌激素E2。PACAP能刺激斑馬魚濾泡細胞中芳香化酶的表達。cAMP類似物，如forskolin和dbCAMP都可以模仿PACAP對芳香化酶的表達。PKA抑製劑 H89，可以完全抑制 PACAP誘導的芳香化酶的表達，這表明PACAP通過cAMP-PKA依賴性途徑調節芳香化酶的表達。由於促性腺激素也使用相同的cAMP-PKA途徑調節芳香化酶的表達，這進一步證實了PACAP是促性腺激素的下游調節者。 / 我們還研究了PACAP對該軸的其他因子調控，以便確定卵巢中是否存在一個有功能的GHRH/PACAP-GH-IGF軸。我們使用斑馬魚原代培養的濾泡細胞作為研究體系進行了一系列的基因調控的研究。PACA可以刺激gh及其受體 ghra和ghrb的表達。此外，它還增加了igf1表達，但對igf2a和igf2b的表達沒有明顯的影響。鑑於之前的工作證明重組zfGH可以刺激igf1的表達，我們有理由相信，在斑馬魚卵巢中存在一個有功能的GHRH/PACAP-GH-IGF軸。PACAP對此軸的調節作用也主要是通過cAMP-PKA途徑。 / 本研究不僅增加了我們對GHRH/PACAP-GH-IGF軸在卵巢中功能的了解，而且還提供了關於魚類生長和繁殖的協調方面有價值的信息，這必將有利於水產養殖。魚脊椎動物中最原始，最多元化的群體，目前的研究結果為其他生物的研究也提供了重要的參考。 / Growth and reproduction are two major physiological processes, which have been proven to be closely related in vertebrates. The process of growth is governed by the brain-pituitary-liver axis involving growth hormone releasing hormone/ pituitary adenylate cyclase-activating polypeptide (GHRH/PACAP), growth hormone (GH) and insulin-like growth factors (IGFs). Interestingly, the expression of all the genes involved in this axis has been reported in the ovary, which indicates that a functional mini-axis might exist in the ovary. In this study, we focus on the characterization of PACAP, the upstream element of the axis, to reveal the existence and functional roles of this intraovarian mini-axis. / PACAP is a neuropeptide originally purified from ovine hypothalamus for its potent activity to stimulate cAMP production. However, its presence and actions have also been demonstrated in various peripheral tissues including the ovary. In the zebrafish, two forms of PACAP (PACAP₃₈-1, adcyap1a; and PACAP₃₈-2, adcyap1b) and three PACAP receptors (PAC1-R, adcyap1r1; VPAC1-R, vipr1 and VPAC2-R, vipr2) were all expressed in the ovary. To provide clues to the importance of the PACAP system in the function of zebrafish ovary, we first investigated the spatial distribution of both PACAP ligands and the three potential receptors in the somatic follicle layer and denuded oocytes. We also analyzed the temporal expression profiles of PACAP ligands and receptors during follicle growth and maturation. Spatiotemporal expression data of PACAP system suggested that PACAP might play dual roles in regulating follicle growth and maturation through different receptors located in different compartments. This hypothesis was further supported by the observation that PACAP promoted maturation of follicle-enclosed oocytes but suppressed spontaneous maturation of denuded oocytes in vitro. / As the expression of PACAP (PACAP₃₈-2) was significantly stimulated by pituitary gonadotropins (hCG) in cultured zebrafish follicle cells, PACAP is therefore likely a downstream mediator or modulator of pituitary gonadotropins in controlling ovarian functions. We illustrated from several aspects to verify our hypothesis. Firstly, we tested the regulation of PACAP on the expression of activin/follistatin system for its well characterized roles in mediating pituitary gonadotropins (hCG). According to our results, PACAP could not only mimic gonadotropin-regulated expression of the activin/follistatin system, but also stimulated activin-mediated oocyte maturation. The same cAMP-dependent signal pathways PACAP and hCG chose towards the differential regulation of activin/follistatin system further confirm PACAP’s role as a mediator or even an amplifier. Secondly, EGF, the ovary-derived growth factor, was administrated to study its effects on PACAP regulated expression of activin/follistatin system. Similar with its influences on hCG regulated genes expression of activin system, EGF could work on its membrane receptors using a MEK pathway to regulate the effects of PACAP. Thirdly, the effect of PACAP on steroidogenesis was also studied. PACAP could stimulate the expression of aromatase, one of the steroidogenic enzymes that could convert testosterone to E2, in cultured zebrafish follicle cells. Its effect on aromatase expression could be mimicked by drugs that increase intracellular cAMP levels such as forskolin and db-cAMP. PACAP induced aromatase expression was totally abolished by a PKA inhibitor H89, which indicated that PACAP worked through a cAMP-PKA dependent pathway to regulate aromatase expression. As gonadotropins also use the same cAMP-PKA pathway to regulate the expression of aromatase, it was further confirmed that PACAP could mediate or amplify the effects of gonadotropins, even in steroidogenesis. / We also studied the regulatory effects of PACAP on other components of this mini-axis to find out whether this hypothetical GHRH/PACAP-GH-IGF axis in the ovary work the same way as the systemic somatotrophic one. We carried out a series of regulatory studies using the primary zebrafish follicle cell culture system. Interestingly, PACAP up-regulated the expression of gh and its receptors ghra and ghrb. In addition, it also increased the expression of igf1 but not igf2a and igf2b. Accompanied with the fact that recombinant zfGH could stimulate the expression of igf1, we have reason to believe that a functional intraovarian axis exited in zebrafish ovary. It seems that the regulatory effects of PACAP on this axis also mediated through a cAMP-PKA pathway. / The present study not only increases our understanding of the GHRH/PACAP- GH-IGFs axis and its actions in the ovary, but also provides valuable information on the coordination of growth and reproduction in fish, which will surely benefit the manipulation of fish growth and breeding in aquaculture. Since fish represent the most primitive and diverse group of vertebrates, the information obtained from the present study will serve as important reference for the studies in other organisms. / 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. / Zhou, Rui. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 127-157). / 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.i / Abstract in Chinese --- p.iv / Acknowledgement --- p.vi / Table of content --- p.viii / List of figures and tables --- p.xiii / Symbols and abbreviation --- p.xvi / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Ovary --- p.1 / Chapter 1.1.1 --- Folliculogenesis --- p.1 / Chapter 1.1.2 --- Steroidogenesis --- p.7 / Chapter 1.1.3 --- Endocrine, paracrine and autocrine network of ovarian follicles --- p.8 / Chapter 1.2 --- GHRH/PACAP-GH-IGF axis --- p.11 / Chapter 1.2.1 --- GHRH/PACAP-GH-IGF axis in growth --- p.11 / Chapter 1.2.2 --- GHRH/PACAP-GH-IGF axis in reproduction --- p.13 / Chapter 1.3 --- Pituitary adenylate cyclase-activating polypeptide (PACAP) family --- p.15 / Chapter 1.3.1 --- PACAP ligands --- p.15 / Chapter 1.3.2 --- PACAP receptors --- p.17 / Chapter 1.3.3 --- Function of PACAP system --- p.21 / Chapter 1.4 --- Objective of present study --- p.23 / Chapter Chapter 2 --- Pituitary Adenylate Cyclase-activating Polypeptide (PACAP) and Its Receptors in the Zebrafish Ovary - Evidence for Potential Dual Roles of PACAP in Controlling Final Oocyte Maturation / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.2 --- Materials and method --- p.30 / Chapter 2.2.1 --- Animals and chemicals --- p.30 / Chapter 2.2.2 --- Isolation of ovarian follicles --- p.31 / Chapter 2.2.3 --- Separation of oocyte and follicle layer --- p.32 / Chapter 2.2.4 --- Follicle incubation and oocyte maturation assay --- p.32 / Chapter 2.2.5 --- Primary follicle cell culture --- p.33 / Chapter 2.2.6 --- RNA extraction and reverse transcription --- p.33 / Chapter 2.2.7 --- Semi- quantitative RT-PCR and real-time qPCR --- p.33 / Chapter 2.2.8 --- Data analysis --- p.34 / Chapter 2.3 --- Results --- p.34 / Chapter 2.3.1 --- Spatial distribution of PACAP system within the follicle --- p.34 / Chapter 2.3.2 --- Temporal expression profiles of the PACAP system during folliculogenesis --- p.35 / Chapter 2.3.3 --- Expression profiles of PACAP system in peri-ovulatory period in vivo --- p.35 / Chapter 2.3.4 --- Expression mark change of the PACAP system during in vivo and in vitro maturation --- p.37 / Chapter 2.3.5 --- Effects of PACAP on final maturation of intact follicles and denuded oocytes --- p.38 / Chapter 2.4 --- Discussion --- p.39 / Chapter Chapter 3 --- PACAP Mimics Pituitary Gonadotropin(s) in Regulating Ovarian Activin/Inhibin/Follistatin System / Chapter 3.1 --- Introduction --- p.54 / Chapter 3.2 --- Materials and method --- p.57 / Chapter 3.2.1 --- Animals and chemicals --- p.57 / Chapter 3.2.2 --- Primary culture of ovarian follicle cells --- p.57 / Chapter 3.2.3 --- Total RNA isolation and reverse transcription --- p.58 / Chapter 3.2.4 --- Real-time polymerase chain reaction --- p.58 / Chapter 3.2.5 --- Isolation and incubation of follicles --- p.59 / Chapter 3.2.6 --- Data analysis --- p.59 / Chapter 3.3 --- Result --- p.61 / Chapter 3.3.1 --- PACAP regulation of the expression of activin/inhibin/follistatin system in cultured zebrafish ovarian follicle cells --- p.61 / Chapter 3.3.2 --- Involvement of protein kinase A (PKA) in the differential regulation of activin subunits and follistatin by PACAP --- p.61 / Chapter 3.3.3 --- Potential role of activin in PACAP-induced oocyte maturation --- p.62 / Chapter 3.3.4 --- Interactive effects of EGF and PACAP on the expression of activin subunits and follistatin in the follicle cells --- p.62 / Chapter 3.3.5 --- Potential involvement of EGF-EGFR signaling in PACAP-regulated expression of activin subunits and follistatin in the follicle cells --- p.62 / Chapter 3.4 --- Discussion --- p.63 / Chapter Chapter 4 --- PACAP Regulation of Ovarian GH-IGF System / Chapter 4.1 --- Introduction --- p.78 / Chapter 4.2 --- Materials and methods --- p.81 / Chapter 4.2.1 --- Animals and chemicals --- p.81 / Chapter 4.2.2 --- Primary culture of ovarian follicle cells --- p.81 / Chapter 4.2.3 --- Total RNA isolation and reverse transcription --- p.82 / Chapter 4.2.4 --- Real-time polymerase chain reaction --- p.82 / Chapter 4.2.5 --- Follicle incubation --- p.82 / Chapter 4.2.6 --- Data analysis --- p.83 / Chapter 4.3 --- Result --- p.83 / Chapter 4.3.1 --- PACAP regulation of the expression of growth hormone and growth hormone receptors in cultured zebrafish ovarian follicle cells --- p.83 / Chapter 4.3.2 --- PACAP regulation of the expression of insulin-like growth factors and their receptors in cultured zebrafish ovarian follicle cells --- p.84 / Chapter 4.3.3 --- Self-regulation of PACAP expression in cultured zebrafish ovarian follicle cells --- p.85 / Chapter 4.3.4 --- Evaluation of protein kinase A (PKA) involvement in the PACAP-regulated expression of GH and IGFs family --- p.85 / Chapter 4.3.5 --- Interactive effects of PACAP and EGF on expression of zebrafish GH-IGF axis in cultured follicle cells --- p.86 / Chapter 4.4 --- Discussion --- p.87 / Chapter Chapter 5 --- PACAP regulation of cytochrome P450 aromatase expression in cultured zebrafish ovarian follicle cells / Chapter 5.1 --- Introduction --- p.103 / Chapter 5.2 --- Materials and methods --- p.106 / Chapter 5.2.1 --- Animals and chemicals --- p.106 / Chapter 5.2.2 --- Primary culture of ovarian follicle cells --- p.106 / Chapter 5.2.3 --- Total RNA isolation and reverse transcription --- p.107 / Chapter 5.2.4 --- Real-time polymerase chain reaction --- p.107 / Chapter 5.2.5 --- Data analysis --- p.108 / Chapter 5.3 --- Results --- p.108 / Chapter 5.3.1 --- PACAP regulation of cyp19a1a expression in cultured zebrafish ovarian follicle cells --- p.108 / Chapter 5.3.2 --- Effects of forskolin and db-cAMP on cyp19a1a expression --- p.109 / Chapter 5.3.3 --- Involvement of protein kinase A (PKA) in the regulation of cyp19a1a expression by PACAP --- p.109 / Chapter 5.4 --- Discussion --- p.109 / Chapter Chapter 6 --- General Discussion / Chapter 6.1 --- Potential roles of PACAP in folliculogenesis --- p.120 / Chapter 6.2 --- PACAP mediates gonadotropins’ signaling through activin/follistatin system --- p.123 / Chapter 6.3 --- PACAP regulation of steroidogenesis --- p.124 / Chapter 6.4 --- PACAP regulation of ovarian PACAP-GH-IGF axis --- p.127 / Reference

Liu Lin. / "January 2006." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 117-135). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

by Henry, Kam Yin Cheung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 148-160). / ZEBRAFISH (BRACHYDANIO RERIO) / ACKNOWLEDGEMENTS / LIST OF CONTENTS / ABSTRACT / ABBREVIATION / Chapter CHAPTER ONE --- INTRODUCTION / Chapter 1.1 --- Transgenic fish --- p.1 / Chapter 1.2 --- Zebrafish --- p.4 / Chapter 1.3 --- The grass carp GH gene and protein / Chapter 1.3.1 --- The genomic sequence --- p.5 / Chapter 1.3.2 --- The cDNA sequence --- p.7 / Chapter 1.3.3 --- The grass carp GH protein --- p.7 / Chapter 1.4 --- Functional aspects of promoter regions / Chapter 1.4.1 --- PEPCK --- p.9 / Chapter 1.4.2 --- RSV-LTR --- p.10 / Chapter 1.4.3 --- hMT-IIA --- p.10 / Chapter 1.4.4 --- MMTV-LTR --- p.11 / Chapter 1.5 --- Eukaryotic gene expression in cultured cells / Chapter 1.5.1 --- COS-7 and HepG2 cells --- p.11 / Chapter 1.5.2 --- Transfection system --- p.12 / Chapter 1.5.3 --- Fate of DNA after transfection --- p.13 / Chapter 1.6 --- Electroporation and microinjection as tools for gene transfer / Chapter 1.6.1 --- Electroporation: Theory and operation --- p.13 / Chapter 1.6.2 --- Microinjection: Design of microinjector --- p.16 / Chapter 1.6.3 --- Fate of DNA after gene transfer in embryos / Transient expression --- p.16 / Stable transformation --- p.17 / Inheredity of transgene --- p.17 / Chapter 1.7 --- The aims of the present study --- p.18 / Chapter CHAPTER TWO --- MATERIALS AND METHODS / Chapter 2.1 --- General techniques / Chapter 2.1.1 --- Electrophoresis of DNA / Chapter 2.1.1.1 --- Agarose gel electrophoresis --- p.19 / Chapter 2.1.1.2 --- PAGE --- p.20 / Chapter 2.1.2 --- Purification of DNA --- p.21 / Chapter 2.1.3 --- Recovery of DNA fragments / Chapter 2.1.3.1 --- Electroelution --- p.22 / Chapter 2.1.3.2 --- Geneclean kit --- p.23 / Chapter 2.1.4 --- Standard recombinant DNA techniques / Chapter 2.1.4.1 --- Dephosphorylation --- p.24 / Chapter 2.1.4.2 --- Kinasing --- p.24 / Chapter 2.1.4.3 --- Ligation --- p.24 / Chapter 2.1.4.4 --- Filling in reaction --- p.25 / Chapter 2.1.4.5 --- Transformation --- p.25 / Chapter 2.1.5 --- Minipreparation of plasmids --- p.26 / Chapter 2.1.6 --- Large preparation of plasmids / Chapter 2.1.6.1 --- Qiagene kit --- p.27 / Chapter 2.1.6.2 --- CsCl density gradient centrifugation --- p.27 / Chapter 2.1.7 --- DNA sequencing --- p.29 / Chapter 2.1.8 --- "Extraction of DNA from embryos, fry and fish" / Method 1 --- p.32 / Method 2 --- p.32 / Chapter 2.1.9 --- Probe labelling / Chapter 2.1.9.1 --- End-labelling --- p.33 / Chapter 2.1.9.2 --- Random priming --- p.33 / Chapter 2.1.10 --- CAT assay --- p.33 / Chapter 2.1.11 --- Polymerase chain reaction(PCR) --- p.35 / Chapter 2.1.12 --- Radioimmunassay(RIA) of FGH --- p.36 / Chapter 2.1.13 --- Dot blotting --- p.38 / Chapter 2.1.14 --- Southern blotting --- p.39 / Chapter 2.2 --- "Linkers, primers and probes" / Chapter 2.2.1 --- Primers --- p.41 / Chapter 2.2.2 --- Linkers --- p.45 / Chapter 2.2.3 --- Probes --- p.47 / Chapter 2.3 --- Construction of chimeric growth hormone genes / Chapter 2.3.1 --- Sources of plasmids --- p.50 / Chapter 2.3.2 --- General principles --- p.50 / Chapter 2.3.3 --- PEPCKgcGHcDNA --- p.51 / Chapter 2.3.4 --- RSVgcGHcDNA --- p.54 / Chapter 2.3.5 --- hMTgcGHcDNAcDNA --- p.56 / Chapter 2.3.6 --- MMTVgcGHcDNA --- p.58 / Chapter 2.3.7 --- "PEPCKgcGH, RSVgcGH and hMTgcGH" --- p.60 / Chapter 2.4 --- Expression of chimeric genes in cultured cells / Chapter 2.4.1 --- Culturing of COS-7 and HepG2 cells --- p.66 / Chapter 2.4.2 --- Expression of chimeric genes in COS-7 and HepG2 cells --- p.67 / Chapter 2.5 --- Zebrafish / Chapter 2.5.1 --- "Culturing, Spawning and hatching" --- p.67 / Chapter 2.6 --- Electroporation and microinjection for gene transfer / Chapter 2.6.1 --- Electroporation / Chapter 2.6.1.1 --- Tuning up electroporation --- p.69 / Chapter 2.6.1.2 --- Evidence of gene transfer by electroporation / Chapter 2.6.1.2.1 --- CAT assay --- p.71 / Chapter 2.6.1.2.2 --- Dot blot --- p.71 / Chapter 2.6.1.2.3 --- PCR and Southern blotting of PCR products --- p.72 / Chapter 2.6.1.2.4 --- Southern blotting of fish total DNA --- p.73 / Chapter 2.6.2 --- Microinjection / Chapter 2.6.2.1 --- Handling of microinjection --- p.74 / Chapter 2.6.2.2 --- Evidence of gene transfer by microinjection / Chapter 2.6.2.2.1 --- CAT assay --- p.75 / Chapter 2.6.2.2.2 --- PCR and Southern blotting of PCR products --- p.75 / Chapter 2.7 --- Phenotypic alteration of fish generated from electroporated eggs / Chapter 2.7.1 --- Electroporation and handling of fish generated from electroporation --- p.75 / Chapter 2.7.2 --- Measurement of phenotypic change in fish generated from electroporation --- p.77 / Chapter 2.8 --- Detection of transgene and expression of exogenous DNA / Chapter 2.8.1 --- Transgene detection --- p.78 / Chapter 2.8.2 --- Expression of exogenous DNA --- p.79 / Chapter CHAPTER THREE --- RESULTS / Chapter 3.1 --- Construction of Chimeric growth hormone genes / Chapter 3.1.1 --- Confirmation of integrity of chimeric genes / PEPCKgcGHcDNA --- p.80 / RSVgcGHcDNA --- p.81 / hMTgcGHcDNA --- p.81 / MMTVgcGHcDNA --- p.81 / "PEPCKgcGH, RSVgcGH and hMTgcGH" --- p.82 / Chapter 3.1.2 --- Yield of chimeric genes from CsCl density gradient centrifugation --- p.82 / Chapter 3.2 --- Chimeric gene expression in COS-7 and HepG2 cells / Chapter 3.2.1 --- Expression of chimeric genes in COS-7 cells --- p.89 / Chapter 3.2.2 --- Expression of chimeric genes in HepG2 cells --- p.93 / Chapter 3.3 --- Transfer of chimeric genes into embryos / Chapter 3.3.1 --- Electroporation / Chapter 3.3.1.1 --- Monitoring of electroporation --- p.94 / Chapter 3.3.1.2 --- Evidence for gene transfer / Chapter 3.3.1.2.1 --- CAT assay --- p.98 / Chapter 3.3.1.2.2 --- Dot blotting --- p.98 / Chapter 3.3.1.2.3 --- PCR and Southern blotting of PCR product --- p.101 / Chapter 3.3.1.2.4 --- Southern blotting of DNA from fish generated from electroporation --- p.106 / Chapter 3.3.2 --- Microinjection / Chapter 3.3.2.1 --- CAT assay --- p.109 / Chapter 3.3.2.2 --- PCR --- p.109 / Chapter 3.4 --- Phenotypic alterations of fish / The first experiment --- p.112 / The second experiment --- p.113 / The third experiment --- p.113 / The fourth experiment --- p.122 / Chapter 3.5 --- Detection of transgene and expression of exogenous DNA / Chapter 3.5.1 --- Transgene --- p.128 / Chapter 3.5.2 --- Possible expression of exogenous DNA --- p.129 / Chapter CHAPTER FOUR --- DISCUSSION / Chapter 4.1 --- Chimeric growth hormone genes --- p.132 / Chapter 4.2 --- Expression of chimeric growth hormone genes in COS-7 and HepG2 cells --- p.134 / Chapter 4.3 --- Transfer of exogenous DNA into embyros --- p.136 / Chapter 4.4 --- Phenotypic alteration of fish developed from electroporated eggs --- p.139 / Chapter 4.5 --- The possible integration and expression of exogenous DNA --- p.143 / Chapter 4.6 --- Conclusions --- p.145 / Chapter 4.7 --- Suggestions for further studies --- p.146 / REFERENCES --- p.148 / Chapter APPENDIX I --- Restriction maps / PEPCKgcGH / PEPCKgcGHcDNA / RSVgcGH / RSVgcGHcDNA / hMTgcGH / hMTgcGHcDNA / MMTVgcGHcDNA / pBH1.2 / pMSG-CAT / pUC19 / hMT-IIA / PBC12BI / RSVCAT / pUC101 / pSEl/S2 / PUCSE2/S1 / pUCS2

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.i / Abstract (in Chinese) --- p.iii / Acknowledgement --- p.v / Table of contents --- p.vi / List of figures and tables --- p.xii / Symbols and abbreviations --- p.xv / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Hypothalamic-pituitary-gonadal axis / Chapter 1.1.1 --- Overview --- p.1 / Chapter 1.1.2 --- Gonadotropin-releasing hormone --- p.1 / Chapter 1.2 --- Folliculogenesis / Chapter 1.2.1 --- Structure of ovarian follicles --- p.2 / Chapter 1.2.2 --- Stages of folliculogenesis --- p.3 / Chapter 1.3 --- Gonadotropins and gonadotropin receptors / Chapter 1.3.1 --- History of teleost gonadotropin and gonadotropin receptors --- p.5 / Chapter 1.3.2 --- Structure --- p.6 / Chapter 1.3.3 --- Function --- p.7 / Chapter 1.3.4 --- GTH-GTHR specificity --- p.9 / Chapter 1.3.5 --- Signal transduction --- p.10 / Chapter 1.3.6 --- Expression profile of gonadotropin receptors --- p.11 / Chapter 1.3.7 --- Regulation of gonadotropin receptors --- p.12 / Chapter 1.4 --- Objectives and significances of the project --- p.14 / Chapter 1.5 --- Figure legends --- p.16 / Chapter 1.6 --- Figures --- p.18 / Chapter 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 Membrane / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Materials and methods / Chapter 2.2.1 --- Animals --- p.25 / Chapter 2.2.2 --- Hormones and chemicals --- p.26 / Chapter 2.2.3 --- Primary follicle cell culture and drug treatment --- p.26 / Chapter 2.2.4 --- Ovarian fragment incubation --- p.27 / Chapter 2.2.5 --- Total RNA extraction and real-time qPCR --- p.27 / Chapter 2.2.6 --- Western blot analysis --- p.27 / Chapter 2.2.7 --- SEAP reporter gene assay --- p.28 / Chapter 2.2.8 --- Data analysis --- p.28 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Differential stimulation of fshr and lhcgr expression in ovarian fragments and follicle cells by estradiol but not testosterone --- p.28 / Chapter 2.3.2 --- Potentiation of follicle cell responsiveness to hCG by E2 pretreatment --- p.30 / Chapter 2.3.4 --- Evidence for transcription but not translation-dependent up-regulation of lhcgr by E2 --- p.30 / Chapter 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.31 / Chapter 2.3.6 --- Evidence for possible localization of estrogen receptors on the plasma membrane --- p.32 / Chapter 2.3.7 --- MAPK dependence of E2 effect on lhcgr expression --- p.32 / Chapter 2.4 --- Discussion --- p.33 / Chapter 2.5 --- Table --- p.38 / Chapter 2.6 --- Figure legends --- p.39 / Chapter 2.7 --- Figures --- p.43 / Chapter Chapter 3 --- Signal Transduction Mechanisms of the Biphasic Estrogen Actions in the Regulation of Gonadotropin Receptors (fshr and lhcgr) in the Zebrafish Ovary / Chapter 3.1 --- Introduction --- p.50 / Chapter 3.2 --- Materials and methods / Chapter 3.2.1 --- Animals --- p.52 / Chapter 3.2.2 --- Hormones and chemicals --- p.52 / Chapter 3.2.3 --- Primary cell culture and drug treatment --- p.52 / Chapter 3.2.4 --- Total RNA extraction and real-time qPCR --- p.52 / Chapter 3.2.5 --- Fractionation of follicle cells --- p.52 / Chapter 3.2.6 --- Western blot analysis --- p.52 / Chapter 3.2.7 --- Statistical analysis --- p.53 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Biphasic roles of cAMP-PKA pathway --- p.53 / Chapter 3.3.2 --- Effects of p38 MAPK inhibition --- p.54 / Chapter 3.3.3 --- Effects of PKC and PI3K inhibition --- p.54 / Chapter 3.4 --- Discussion --- p.55 / Chapter 3.5 --- Figure legends --- p.59 / Chapter 3.6 --- Figures --- p.61 / Chapter 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 pathway / Chapter 4.1 --- Introduction --- p.66 / Chapter 4.2 --- Materials and methods / Chapter 4.2.1 --- Animals --- p.69 / Chapter 4.2.2 --- Hormones and chemicals --- p.69 / Chapter 4.2.3 --- Primary cell culture and drug treatment --- p.69 / Chapter 4.2.4 --- Total RNA extraction and real-time qPCR --- p.69 / Chapter 4.2.5 --- Statistical analysis --- p.69 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Down-regulation of fshr and lhcgr by hCG --- p.69 / Chapter 4.3.2 --- Differential regulation of fshr and lhcgr by PACAP --- p.70 / Chapter 4.3.3 --- Inhibition of E2-regulated fshr and lhcgr expression by hCG --- p.71 / Chapter 4.3.4 --- Suppressive effects of PACAP on E2-induced fshr and lhcgr expression --- p.71 / Chapter 4.3.5 --- Role of cAMP in hCG and PACAP actions --- p.72 / Chapter 4.4 --- Discussion --- p.73 / Chapter 4.5 --- Figure legends --- p.78 / Chapter 4.6 --- Figures --- p.80 / Chapter 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.85 / Chapter 5.2 --- Materials and methods / Chapter 5.2.1 --- Animals --- p.88 / Chapter 5.2.2 --- Hormones and chemicals --- p.88 / Chapter 5.2.3 --- Primary cell culture and drug treatment --- p.88 / Chapter 5.2.4 --- Total RNA extraction and real-time qPCR --- p.88 / Chapter 5.2.5 --- Statistical analysis --- p.88 / Chapter 5.3 --- Results / Chapter 5.3.1 --- Biphasic down-regulation of lhcgr by EGF --- p.89 / Chapter 5.3.2 --- Evidence for EGFR involvement --- p.89 / Chapter 5.3.3 --- Minor role of MEK-MAPK3/1 pathway in the EGF effect on lhcgr expression --- p.90 / Chapter 5.3.4 --- Up-regulation of fshr by IGF-I --- p.90 / Chapter 5.3.5 --- Evidence for IGF-IR involvement --- p.91 / Chapter 5.3.6 --- Role of PI3K-Akt pathway in IGF-I action --- p.91 / Chapter 5.3.7 --- Role of EGF and EGFR in E2-induced GTHR expression --- p.91 / Chapter 5.3.8 --- Role of IGF-I and IGF-IR in E2-induced GTHR expression --- p.91 / Chapter 5.4 --- Discussion --- p.92 / Chapter 5.5 --- Figure legends --- p.98 / Chapter 5.6 --- Figures --- p.100 / Chapter Chapter 6 --- Regulation of estrogen receptor subtypes (esr1, esr2a and esr2b): a possible mechanism to modulate estradiol-stimulated lhcgr expression in the zebrafish ovary / Chapter 6.1 --- Introduction --- p.107 / Chapter 6.2 --- Materials and methods / Chapter 6.2.1 --- Animals --- p.110 / Chapter 6.2.2 --- Hormones and chemicals --- p.110 / Chapter 6.2.3 --- Staging ovarian follicles --- p.110 / Chapter 6.2.4 --- Primary cell culture and drug treatment --- p.110 / Chapter 6.2.5 --- Total RNA extraction and real-time qPCR --- p.110 / Chapter 6.2.6 --- Statistical analysis --- p.111 / Chapter 6.3 --- Results / Chapter 6.3.1 --- Expression profiles of estrogen receptors (ERs) in zebrafish folliculogenesis --- p.111 / Chapter 6.3.2 --- Homologous regulation of nERs by E2 --- p.111 / Chapter 6.3.3 --- Regulation of nERs by endocrine hormones (hCG and PACAP) --- p.112 / Chapter 6.3.4 --- Regulation of nERs by ovarian paracrine growth factors (EGF and IGF-I) --- p.112 / Chapter 6.3.5 --- Role of cAMP in nER regulation --- p.113 / Chapter 6.3.6 --- Role of PKA in nER regulation --- p.113 / Chapter 6.4 --- Discussion --- p.114 / Chapter 6.5 --- Figure legends --- p.119 / Chapter 6.6 --- Figures --- p.121 / Chapter Chapter 7 --- Estrogenic Action Mechanisms of Bisphenol A / Chapter 7.1 --- Introduction --- p.127 / Chapter 7.2 --- Materials and methods / Chapter 7.2.1 --- Animals --- p.129 / Chapter 7.2.2 --- Hormones and chemicals --- p.129 / Chapter 7.2.3 --- Primary cell culture and drug treatment --- p.129 / Chapter 7.2.4 --- Total RNA extraction and real-time qPCR --- p.129 / Chapter 7.2.5 --- Statistical analysis --- p.130 / Chapter 7.3 --- Results / Chapter 7.3.1 --- Expression of fshr and lhcgr interfered by BPA --- p.130 / Chapter 7.3.2 --- Signaling mechanism of BPA-induced lhcgr up-regulation --- p.130 / Chapter 7.3.3 --- Dependence of transcription and translation in BPA-induced lhcgr expression --- p.131 / Chapter 7.3.4 --- Evidence for the involvement of nuclear estrogen receptors in the BPA actions --- p.131 / Chapter 7.3.5 --- Interference on E2-induced lhcgr expression by BPA --- p.131 / Chapter 7.4 --- Discussion --- p.132 / Chapter 7.5 --- Figure legends --- p.136 / Chapter 7.6 --- Figures --- p.138 / Chapter Chapter 8: --- General Discussion / Chapter 8.1 --- Estradiol as a differential regulator of gonadotropin receptors --- p.143 / Chapter 8.2 --- Conserved role of estradiol with differential action mechanisms in lhcgr regulation of mammals and teleosts --- p.144 / Chapter 8.3 --- Involvement of classical estrogen receptors that are likely located on the plasma membrane --- p.145 / Chapter 8.4 --- Biphasic response of lhcgr to estradiol and the underlying signal transduction mechanisms --- p.145 / Chapter 8.5 --- Desensitization of follicle cells to gonadotropins by hCG --- p.146 / Chapter 8.6 --- Paracrine control of gonadotropin receptors by ovarian growth factors --- p.147 / Chapter 8.7 --- Interaction of the estrogen action with other endocrine and paracrine signals --- p.148 / Chapter 8.8 --- Action mechanism studies of an endocrine-disrupting chemical: bisphenol A --- p.150 / Chapter 8.9 --- Conclusion --- p.151 / Chapter 8.10 --- Figure legends --- p.153 / Chapter 8.11 --- Figures --- p.155 / References --- p.159

As a functional unit involved in both maintaining endocrine homeostasis and also producing mature eggs, the ovary plays a central role in female reproduction. The development and function of the ovarian follicles are controlled by gonadotropins released from the pituitary. It is widely accepted that the action of gonadotropins on ovarian follicles is mediated by paracrine/autocrine factors produced by the somatic cells surrounding the oocyte. Increasing evidence indicates that the Igf system is involved in mediating the action of gonadotropins in the ovary. Previously, we identified a gonad-specific Igf subtype (Igf3) distinct from Igf1 and Igf2. This fmding further highlights the importance of the Igf system in the fish ovary. In this thesis, efforts were made to understand the role of the Igf system in ovary using zebrafish as the model organism, and attention was focused on Igf3. / Because the expression of Igf3 is correlated with the LH receptor in zebrafish follicles, the regulation of igf3 by gonadotropins was subsequently studied in the ovary. The expression of igf3 was significantly up-regulated in both ovarian fragments and isolated follicles upon treatment with hCG in dose- and time-dependent manners. Treatment with 8-Br-cAMP or IBMX mimicked the effects of hCG on the expression of igf3 in follicles of different stages. / Four Igfs are present in zebrafish, and our results show that all four igfs are expressed in the ovary of zebrafish and exhibit the differential expression profiles during folliculogenesis. Using a primary culture of zebrafish follicle cells, we demonstrated that hCG stimulated igf2b and igf3 expression but suppressed igf2a expression. Moreover, the effect of gonadotropin could be mimicked by IBMX, which increased the intracellular levels of cAMP, suggesting the possible involvement of cAMP in the gonadotropin-based regulation and differential expression of igf2a, igf2b and igf3. These results also show that the Igf3 is the Igf subtype most sensitive to gonadatropin and cAMP. / In addition, the expression patterns of igf1, igf2a, igf2b, igf3, igf1ra and igf1rb were also studied during zebrafish embryogenesis. The unique temporal and spatial expression patterns of igf1, igf2a, igf2b, igf3, igf1ra and igf1rb were revealed by both real-time PCR and whole mount in situ hybridization, the results suggest divergent functions for these Igfs in early zebrafish development. / Taken together, the present studies provide substantial information about the Igf system, especially that of Igf3 in the zebrafish ovary. Data were gathered regarding Igf3 expression, regulation and functions, which is not only helpful for the understanding of the role of the Igf system in fish reproduction, but also contributes toward uncovering the ovarian signaling network involved in oocyte maturation across vertebrates. This study of igfs gene expression provides direct information to the study of Igf signaling in zebrafish. / To study the function of Igf3, bioactive recombinant Igf3 proteins were prepared using a bacterial expression system. Incubation of follicles with recombinant zebrafish Igf3 significantly enhanced oocyte maturation in time-, dose- and stage-dependent manners. The potential mechanisms of Igf3-induced oocyte maturation were then investigated. Igf3 stimulated oocyte maturation via a steroid-independent manner. Igf3 induced oocyte maturation through Igf1rs and the PI3 kinase, PDE3 and MAP kinase were necessary for Igf3-mediated oocyte maturation in zebrafish. / We first examined the gene expression patterns of Igf3 in the ovary. The igf3 gene in zebrafish was found to be alternatively spliced into two transcripts, with transcript variant 1 exclusively expressed in the gonads and transcript variant 2 only expressed during early development. Using specific antibodies developed for zebrafish Igf3, both the prepropeptide and the mature peptide forms of Igf3 were found to be predominantly expressed in the zebrafish ovary. Real-time PCR and in situ hybridization revealed that igf3 mRNA levels were relatively low in the early follicles but significantly increased after the mid vitellogenic stage (midstage III) and were high in the full grown follicles. In the full grown follicles, igf3 mRNA was detected primarily in the somatic follicular cells, with a low level of expression in the oocytes. Igf3 immunoreactivity was confined to the follicular cells alone. / Li, Jianzhen. / Advisers: Hui Zhao; Hon Ki Christopher Cheng. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 122-150). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.