O enriquecimento ambiental oferece estímulos sensoriais, sociais e cognitivos possibilitando ao animal bem-estar e comportamentos mais próximos ao seu natural. Alterações ambientais podem gerar mudanças de expressão gênica, bem como de comportamento. Assim, o primeiro objetivo deste trabalho foi avaliar a influência de diferentes protocolos de enriquecimento ambiental sobre o perfil comportamental do peixe-zebra (Danio rerio) adulto. Utilizando pedras ao fundo do aquário, plantas artificiais e os animais em cardume, foram montados três protocolos de enriquecimento ambiental. O “Protocolo 1” foi desenhado para avaliar se um ambiente enriquecido com os componentes básicos de complexidade por sete dias já induziria alterações no comportamento do peixe-zebra. Os resultados obtidos com o teste de open tank demonstraram que os animais do grupo ambiente enriquecido (AE) apresentaram um perfil locomotor diferenciado, com redução de distância percorrida total e velocidade média, quando comparado com animais do grupo ambiente padrão (AP). Entretanto, esta diferença se deve a uma instabilidade comportamental apresentada pelo grupo AP através do aumento de distância percorrida no fundo do aquário. Não foram observadas alterações no comportamento tipo ansiedade e preferência social entre os grupos. Desse modo, desenhamos o “Protocolo 2” a fim de avaliar se a reposta comportamental se manteria durante uma exposição mais longa (quatorze dias) ao enriquecimento ambiental. Entretanto, a diferença no perfil locomotor entre os grupos não foi apresentada neste protocolo. Com esses resultados, demonstrou-se a intensidade do estresse provocado pela simples troca entre aquários idênticos utilizados normalmente em biotérios, necessitando uma ambientação mínima de duas semanas no aquário moradia antes da realização de testes comportamentais Por último, realizamos a avaliação do “Protocolo 3”, com o intuito de estudar se a renovação de itens da complexidade do enriquecimento ambiental dentro do período quatorze dias teria alteração nos resultados já observados. Os animais do grupo AE não apresentaram diferença no perfil comportamental geral mesmo com a inserção de novos objetos, entretanto, houve uma redução de distância percorrida no fundo do aparato. Em peixe-zebra, estudos já demonstraram a indução da plasticidade neuronal e aumento na proliferação neuronal quando submetidos a protocolos de enriquecimento. No modelo de roedores, muitos dos efeitos do enriquecimento ambiental são relacionados ao aumento de neurogênese adulta e de ramificação dendrítica, bem como alteração da funcionalidade de alguns sistemas de neurotransmissores, como o glutamatérgico. Assim, a segunda parte dessa dissertação teve como objetivo a análise da expressão proteica de um dos transportadores vesiculares de glutamato, o vGluT2, em animais expostos aos protocolos citados anteriormente Os vGluTs são expressos em regiões dendríticas dos neurônios e quanto maior sua expressão proteica, maior é a densidade de vesículas sinápticas presentes. Os dados obtidos demonstram que essa resposta é relacionada a consolidação e habituação dos peixes a essa condição por um período mais prolongado no protocolo de enriquecimento ambiental (Protocolo 2). Neste trabalho, foi demonstrado que o protocolo de enriquecimento ambiental altera a resposta comportamental do peixe-zebra adulto, mantendo-a mais regular do que a dos controles e isso, provavelmente, é decorrente da estimulação de processos direcionados a resposta ao estresse da novidade, bem como de vias relacionadas com a adaptabilidade. Ademais, demonstramos que a habituação por um protocolo mais longo à condição de um ambiente enriquecido é mais importante para desencadear o aumento de expressão proteica de vGluT2 em cérebro de peixe-zebra. / Environmental enrichment offers social, sensory and cognitive stimuli making possible animal welfare and behavior closer to its natural. Environmental alterations can generate changes in gene expression as well as in behavior. Thus, first aim of this study was to evaluate the influence of different environmental enrichment protocols in adult zebrafish’s (Danio rerio) behavioral profile. Considering items more commonly described in the literature (stones at tank bottom, artificial plants and shoal), we designed three enriched environment protocols. “Protocol 1” was developed to evaluate if an enriched environment with basics components of complexity for seven days already induce behavior alterations in zebrafish. Results obtained with open tank test demonstrated that animals submitted to environmental enrichment (EE) presented differentiated locomotor profile by a reduction of total distance travelled and average speed when compared with animals in the standard environment (SE). However, this difference was due to a behavioral instability presented by animals of SE group reflected in an increase of distance travelled in last two minutes of the test. There were not alterations in anxiety-like behavior and social preference between the groups. In this way, the “Protocol 2” was designed to evaluate whether these behavioral responses in open tank test remain in a longer exposure (fourteen days) to environmental enrichment. However, the difference in locomotor profile was not significant between groups. With the results of these two first protocols, the intensity of stress provoked by a simple change of equal tanks, normally utilized in facilities, was demonstrated. This also presented the necessity of a minimum of two weeks of habituation in home tank, before conducting any behavioral test Finally, we realized the evaluation of “Protocol 3” in order to study if a renovation of enriched environment items of complexity within the period of fourteen days would change results already observed. Animals of EE group showed no difference in general behavioral profile even with the insertion of new objects in environment, similarly to that observed in Protocol 2, however a reduction of distance travelled was presented by these animals, reflecting a little of Protocol 1. In zebrafish, some studies have already demonstrated the induction of neuronal plasticity and increase of neuronal proliferation when animals were submitted to enriched environment protocols. In rodent models, many of effects of environmental enrichment are related to increased adult neurogenesis and dendritic branching, as well as alteration of functionality of some neurotransmitter system was known – like glutamatergic. Thus, second part of this dissertation had as objective the analyses of protein expression of one of the vesicular glutamate transporter (vGluT), the vGluT2, in those three enriched environment protocols cited previously by Western Blotting technique The vGluTs were expressed in neuronal dendritic regions and more its expression, more density of synaptic vesicles presents in analyzed region. Data obtained demonstrated that the increase of immunocontent of vGluT2 is related with consolidation and habituation of fish to this condition for a longer period in environmental enrichment (Protocol 2). In this study, there was demonstrated that environmental enrichment alters the adult zebrafish’s behavioral response, keeping it more regular than the control group (SE) and, probably, this is due to stimulation of process directed to stress response of novelty as well as of pathways related to adaptability. In addition, we have demonstrated that habituation to the condition by a longer protocol of enriched environment is more important to trigger the increase of vGluT2 protein expression in total brain of adult zebrafish.

Kwok Hin-Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 84-100). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iv / Acknowledgement --- p.vi / Table of contents --- p.vii / List of figures --- p.xi / List of tables --- p.xiv / Symbols and abbreviations --- p.xv / List of fish names mentioned in the thesis --- p.xviii / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Gonadotropins / Chapter 1.1.1 --- Structure --- p.1 / Chapter 1.1.2 --- Function --- p.4 / Chapter 1.2 --- Gonadotropin receptor / Chapter 1.2.1 --- Structure --- p.5 / Chapter 1.2.2 --- Expression --- p.7 / Chapter 1.2.3 --- Signaling / Chapter 1.2.3.1 --- cAMP-mediated pathway --- p.7 / Chapter 1.2.3.2 --- Phospholipase C-mediated pathway --- p.9 / Chapter 1.2.4 --- Regulation of expression --- p.12 / Chapter 1.2.5 --- Desensitization of receptors / Chapter 1.2.5.1 --- Uncoupling --- p.13 / Chapter 1.2.5.2 --- Internalization --- p.13 / Chapter 1.3 --- Structure of ovarian follicles --- p.14 / Chapter 1.4 --- The project objectives and long-term significance --- p.16 / Chapter Chapter 2 --- Cloning and Characterization of Zebrafish Follicle-stimulating Hormone (FSH) and Luteinizing Hormone (LH) Receptors ´ؤ Evidence for Distinct Functions of FSH and LH in Follicle Development / Chapter 2.1 --- Introduction --- p.19 / Chapter 2.2 --- Materials and Methods / Chapter 2.2.1 --- Animals and chemicals --- p.22 / Chapter 2.2.2 --- Isolation of total RNA --- p.22 / Chapter 2.2.3 --- Cloning of zebrafish FSHR (zfFSHR) and LHR (zfLHR) cDNA fragments from the zebrafish ovary --- p.23 / Chapter 2.2.4 --- Rapid amplification of 5´ةcDNA ends (5'-RACE) and full-length cDNA --- p.24 / Chapter 2.2.5 --- Isolation of ovarian follicles --- p.25 / Chapter 2.2.6 --- Sampling of the ovaries from sexually immature zebrafish --- p.25 / Chapter 2.2.7 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.25 / Chapter 2.2.8 --- Construction of expression plasmids --- p.26 / Chapter 2.2.9 --- Transient transfection and reporter gene assay --- p.27 / Chapter 2.2.10 --- Establishment and characterization of stable zfFSHR or zfLHR-expressing cell lines --- p.28 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Cloning of FSHR and LHR cDNA from the zebrafish ovary --- p.29 / Chapter 2.3.2 --- Functional characterization of zfFSHR and zfLHR --- p.30 / Chapter 2.3.3 --- Expression of zfFSHR and zfLHR during sexual maturation --- p.31 / Chapter 2.3.4 --- Stage-dependent expression of zfFSHR and zfLHR in the ovarian follicles --- p.32 / Chapter 2.4 --- Discussion --- p.33 / Chapter Chapter 3 --- Down-regulation of FSHR and LHR Expression in the Zebrafish Follicle Ceils by Gonadotropin (hCG) and Its Sigaling Mechanism / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Materials and Methods / Chapter 3.2.1 --- Animals --- p.54 / Chapter 3.2.2 --- Chemicals and hormones --- p.54 / Chapter 3.2.3 --- Primary follicle cell culture --- p.55 / Chapter 3.2.4 --- Total RNA isolation --- p.55 / Chapter 3.2.5 --- "Validation of semi-quantitative RT-PCR assays for FSHR, LHR and GAPDH" --- p.56 / Chapter 3.2.6 --- Data analysis --- p.57 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Validation of semi-quantitative RT-PCR assays --- p.57 / Chapter 3.3.2 --- Gonadotropin regulation of FSHR and LHR expression in cultured zebrafish ovarian follicle cells --- p.58 / Chapter 3.3.3 --- Effect of db-cAMP and forskolin on FSHR and LHR expression --- p.59 / Chapter 3.3.4 --- Effects of H89 on hCG-induced suppression of FSHR and LHR expression --- p.60 / Chapter 3.4 --- Discussion --- p.60 / Chapter Chapter 4 --- General Discussion --- p.75 / Chapter 4.1 --- Cloning of zebrafish FSHR and LHR cDNAs and demonstration of receptor specificity --- p.77 / Chapter 4.2 --- Evidence for the differential expression of FSHR and LHR in the zebrafish ovarian and follicle development --- p.78 / Chapter 4.3 --- Down-regulation of FSHR and LHR expression in the zebrafish follicle cells by gonadotropin (hCG) --- p.79 / Chapter 4.4 --- Future research direction --- p.80 / References --- p.84

So Wai-Kin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 100-127). / Abstracts in English and Chinese. / Acknowledgement --- p.I / Abstract (in English) --- p.II / Abstract (in Chinese) --- p.IV / Table of contents --- p.VI / List of Figures --- p.X / Symbols and Abbreviations --- p.XII / List of fish names mentioned in the thesis --- p.XIV / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Pituitary --- p.1 / Chapter 1.2 --- Gonadotropins --- p.1 / Chapter 1.2.1 --- Structure --- p.2 / Chapter 1.2.2 --- Signaling --- p.3 / Chapter 1.2.3 --- Expression --- p.5 / Chapter 1.2.4 --- Functions --- p.7 / Chapter 1.2.4.1 --- Gonadotropin actions on gametogenesis --- p.7 / Chapter 1.2.4.2 --- Gonadotropin actions on steroidogenesis --- p.8 / Chapter 1.2.5 --- Regulation --- p.9 / Chapter 1.2.5.1 --- Neuroendocrine control --- p.10 / Chapter 1.2.5.1.1 --- Gonadotropin-releasing hormone (GnRH) --- p.10 / Chapter 1.2.5.1.2 --- Dopamine (DA) --- p.12 / Chapter 1.2.5.2 --- Gonadal steroid feedback --- p.12 / Chapter 1.2.5.2.1 --- Positive feedback --- p.13 / Chapter 1.2.5.2.2 --- Negative feedback --- p.14 / Chapter 1.2.5.3 --- Paracrine regulators within pituitary --- p.15 / Chapter 1.3 --- Objectives of the present study --- p.16 / Chapter Chapter 2 --- "Molecular Cloning and Functional Characterization of Zebrafish FSHβ, LHβ and GTHα subunits" / Chapter 2.1 --- Introduction --- p.19 / Chapter 2.2 --- Materials and methods --- p.21 / Chapter 2.2.1 --- Chemicals --- p.21 / Chapter 2.2.2 --- Animals --- p.21 / Chapter 2.2.3 --- Genomic DNA isolation --- p.22 / Chapter 2.2.4 --- Total RNA isolation --- p.22 / Chapter 2.2.5 --- Cloning of zebrafish FSHp，LHβ and GTHa fragments --- p.23 / Chapter 2.2.5.1 --- LHβ and GTHα --- p.23 / Chapter 2.2.5.2 --- FSHβ --- p.23 / Chapter 2.2.6 --- "5'- and 3'-RACE of zebrafish FSHp, LHβ and GTHα subunits" --- p.24 / Chapter 2.2.7 --- Construction of expression constructs --- p.25 / Chapter 2.2.8 --- Cell culture and transfection of Flp-In´ёØ CHO cell --- p.26 / Chapter 2.2.9 --- Recombinant production of zebrafish FSH and LH --- p.27 / Chapter 2.2.10 --- Reverse transcription-polymerase chain reaction (RT-PCR) analysis --- p.27 / Chapter 2.2.11 --- Northern blot hybridization --- p.28 / Chapter 2.2.12 --- SEAP reporter gene assay --- p.28 / Chapter 2.2.13 --- Data analysis --- p.29 / Chapter 2.3 --- Results --- p.30 / Chapter 2.3.1 --- "Cloning of zebrafish FSHβ, LHβ and GTHα subunits" --- p.30 / Chapter 2.3.2 --- "Expression of zebrafish FSHp, LHβ and GTHα in the zebrafish pituitary" --- p.31 / Chapter 2.3.3 --- Recombinant production of zebrafish FSH and LH --- p.32 / Chapter 2.3.4 --- Functional analysis of zebrafish FSH and LH --- p.33 / Chapter 2.4 --- Discussion --- p.34 / Chapter Chapter 3 --- "Spatial Expression Patterns of Zebrafish FSHβ, LHβ and GTHα Subunits in the Pituitary and Their Temporal Expression Profiles during Sexual Maturation and Ovulatory Cycle" / Chapter 3.1 --- Introduction --- p.58 / Chapter 3.2 --- Materials and methods --- p.61 / Chapter 3.2.1 --- Chemicals --- p.61 / Chapter 3.2.2. --- Animals --- p.62 / Chapter 3.2.3 --- Total RNA isolation from zebrafish pituitaries and reverse transcription --- p.62 / Chapter 3.2.4 --- Validation of RT-PCR on single pituitary --- p.63 / Chapter 3.2.5 --- Real-time PCR --- p.64 / Chapter 3.2.6 --- Tissue preparation for in situ hybridization --- p.64 / Chapter 3.2.7 --- In situ hybridization --- p.65 / Chapter 3.2.8 --- Data analysis --- p.66 / Chapter 3.3 --- Results --- p.66 / Chapter 3.3.1 --- "PCR amplification of FSHβ, LHβ and GTHα and GAPDH in single zebrafish pituitary" --- p.67 / Chapter 3.3.2 --- "Establishement of real-time RT-PCR for zebrafish FSHβ, LHβ and GTHa and GAPDH" --- p.67 / Chapter 3.3.3 --- "Temporal expression profiles of zebrafish FSHβ, LHβ and GTHα subunits during sexual maturation" --- p.67 / Chapter 3.3.4 --- "Temporal expression profiles of zebrafish FSHp, LHβ and GTHα subunits during ovulatory cycle" --- p.68 / Chapter 3.3.5 --- "In situ hybridization of zebrafish FSHβ, LHβ and GTHα" --- p.69 / Chapter 3.4 --- Discussion --- p.70 / Chapter Chapter 4 --- General Discussion / Chapter 4.1 --- Cloning of zebrafish gonadotropin subunit cDNAs --- p.91 / Chapter 4.2 --- Bioactivity and receptor specificity of recombinant zebrafish FSH and LH --- p.91 / Chapter 4.3 --- Expression of gonadotropin subunits during zebrafish sexual maturation and ovulatory cycle --- p.92 / Chapter 4.4 --- "Localization of FSHβ, LHβ and GTHα subunits in zebrafish pituitary" --- p.93 / Chapter 4.5 --- Contributions of the present study --- p.94 / Chapter 4.6 --- Future prospects --- p.95 / References --- p.100

Forkhead domain基因家族編碼了很多對於胚胎發育至關重要的轉錄因子，而Foxp4則屬於p-subtype forkhead轉錄因子其中一員。Foxp4在胚胎發育期間的表達十分活躍，在發育中的腦部的不同地方表達，但其於中樞神經系統發育中的調控角色並不清楚。Foxp4基因剔除小鼠在出生前死於心臟的缺陷表型(心二分支) ，在此時間段，腦部的發育才剛剛開始，因此我們無法利用Foxp4基因剔除小鼠作為研究中樞神經系統發育的動物模型。最近，我們的團隊利用小腦組織培養技術及siRNA發佈的研究顯示，Foxp4在小鼠小腦中的蒲金氏細胞(Purkinje cell)中擔當著重要的維持作用。這項研究結果加深了我們對研究Foxp4在中樞神經系統發育中的調控角色的決心。 / 本論文旨在利用斑馬魚作為實驗模型，研究foxp4在斑馬魚中樞神經系統發育中的表達及調控角色。RT-PCR結果顯示foxp4在斑馬魚發育中的bud stage開始表達，並在及後的階段維持其表達水平。利用原位雜交技術 (whole mount in-situ hybridization)，我們發現foxp4表達的地區主要集中於發育中的腦部。在成年斑馬魚中，foxp4表達在不同組織和器官，包括腦部，眼睛和心臟。成年斑馬魚腦部切片原位雜交 (sectioned in-situ hybridization)則顯示，foxp4在小腦的蒲金氏細胞和視頂蓋(optic tectum)的periventricular gray zone表達。 / 為了進一步探究foxp4對於胚胎發育過程中的功能，我們利用微注射技術，把反義嗎啉 (morpholino) MO1注射到斑馬魚胚胎中，大幅度抑制foxp4的表達水平。胚胎受精後48小時，MO1注入的胚胎顯示出第四腦室腦積水的缺陷表型。組織學分析顯示，第四腦室以下的延髓被壓縮致形態異常。此外，利用原位雜交技術及不同的分子標記，我們發現胚胎的中後腦邊界也會出現輕度畸形，而後腦的神經元數量及排列亦受到影響。 / 本項研究展示foxp4在胚胎中樞神經系統的發展的重要性，亦提供了新的見解。我們認為foxp4可能是調控腦室發育的重要成員，但在此方面與foxp4相關的分子機制仍須作更深入的研究。 / The forkhead domain gene family encodes a large group of transcription factors that play essential roles in development. Foxp4 is one of the members in the Foxp subfamily that expressed in different parts of developing central nervous system (CNS) and its function is less characterized. Previous study on Foxp4-knockout mice resulted in early embryonic lethality due to defective heart tube development that hindered the functional study of Foxp4 in CNS development. Recently, our laboratory reported that Foxp4 functions as a maintenance role in the Purkinje cell in the mouse cerebellum. Nevertheless, the role of foxp4 in CNS development was still unclear. / In this study, we used zebrafish as a model to study the expression pattern and functional study of foxp4 in the developing CNS. RT-PCR analysis showed that foxp4 transcript was expressed at the bud stage and maintained in the later embryonic stages. Whole-mount in-situ hybridization showed that foxp4 expressed in the cephalic region during embryonic development. In adult zebrafish, foxp4 expresses in different tissues and organs including brain, eye and heart. Sectioned in-situ hybridization of the adult zebrafish brain showed that foxp4 was specifically expressed in the Purkinje cell and the periventricular gray zone of optic tectum. / To further investigate the function of foxp4 during embryonic development, we injected antisense morpholino, MO1 into the zebrafish embryo to knockdown foxp4. By 48 hour post fertilization (hpf), MO1-injected embryos displayed hydrocephalus in the 4th ventricle. Histological analysis revealed that the medulla oblongata below the 4th ventricle was compressed by the edema resulting in abnormal morphology of medulla oblongata in the MO1-injected morphant. In addition, a mild malformation of the mid-hindbrain boundary, disrupted hindbrain patterning was observed in MO1-injected morphant. / Our findings provide new insight into the function of foxp4 in embryonic CNS development. We suggested that foxp4 may be essential in regulating the brain ventricle development while the molecular mechanism underlying the functional role of foxp4 requires further investigation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wong, Wai Kei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 92-102). / Abstracts also in Chinese. / Abstract --- p.iii / 摘要 --- p.v / Acknowledgement --- p.vii / Figure and table list --- p.xi / Abbreviation --- p.xii / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Zebrafish as a developmental model --- p.1 / Chapter 1.2 --- Zebrafish development with highlights --- p.3 / Chapter 1.2.1 --- CNS development --- p.3 / Chapter 1.3 --- Forkhead domain gene in development --- p.5 / Chapter 1.3.1 --- History of forkhead domain gene --- p.5 / Chapter 1.3.2 --- Functional roles of forkhead domain genes in development --- p.6 / Chapter 1.4 --- Foxp subfamily --- p.8 / Chapter 1.4.1 --- Diverse functions of Foxp1, 2, 3 and 4 --- p.8 / Chapter 1.4.2 --- Relationship between Foxp subfamily members --- p.10 / Chapter 1.5 --- Foxp4 --- p.11 / Chapter 1.5.1 --- Genomic organization and protein structure of mFoxp4 --- p.11 / Chapter 1.5.2 --- Previous studies of mFoxp4 --- p.14 / Chapter 1.5.3 --- Foxp4 studies in other model organisms --- p.14 / Chapter 1.5.3.1 --- Rat --- p.15 / Chapter 1.5.3.2 --- Xenopus --- p.16 / Chapter 1.5.3.3 --- C. elegans --- p.16 / Chapter 1.5.4 --- Zebrafish foxp4 --- p.17 / Chapter 1.5.5.1 --- Genomic organization and protein structure of foxp4 --- p.17 / Chapter 1.5.5.2 --- Sequence alignment of foxp4 with other models --- p.19 / Chapter 1.6 --- Hypothesis, aim and strategy of the study --- p.22 / Chapter Chapter 2 --- Expression of foxp4 in zebrafish embryo and adult zebrafish brain / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Materials and methods --- p.25 / Chapter 2.2.1 --- Animals --- p.25 / Chapter 2.2.2 --- Materials --- p.26 / Chapter 2.2.3 --- Semi-quantitative PCR --- p.35 / Chapter 2.2.3.1 --- cDNA of zebrafish embryo --- p.35 / Chapter 2.2.3.2 --- Isolation of adult zebrafish organs --- p.36 / Chapter 2.2.3.3 --- RNA extraction and reverse transcription --- p.36 / Chapter 2.2.3.4 --- Polymerase chain reaction --- p.37 / Chapter 2.2.4 --- Subcloning of DNA fragment / Chapter 2.2.4.1 --- Preparation of cloning vectors --- p.40 / Chapter 2.2.4.2 --- Subcloning of DNA fragments --- p.40 / Chapter 2.2.4.3 --- Transformation of DNA into competent cells --- p.40 / Chapter 2.2.4.4 --- Preparation of recombinant plasmid DNA --- p.41 / Chapter 2.2.5 --- Whole mount in-situ hybridization of zebrafish embryo --- p.45 / Chapter 2.2.5.1 --- Preparation of equipment --- p.45 / Chapter 2.2.5.2 --- Preparation of zebrafish embryos --- p.45 / Chapter 2.2.5.3 --- Preparation of RNA probe --- p.46 / Chapter 2.2.5.4 --- Whole-mount in-situ hybridization --- p.48 / Chapter 2.2.6 --- Sectioned in-situ hybridization of adult zebrafish brain --- p.49 / Chapter 2.2.6.1 --- Histology of adult zebrafish brain --- p.49 / Chapter 2.2.6.2 --- Sectioned in-situ hybridization --- p.50 / Chapter 2.3 --- Results --- p.51 / Chapter 2.3.1 --- Expression profile of foxp4 in different stages of zebrafish embryo --- p.51 / Chapter 2.3.2 --- Expression pattern of foxp4 in different stages of zebrafish embryo --- p.54 / Chapter 2.3.3 --- Expression profile of foxp4 in different zebrafish organs and tissues --- p.57 / Chapter 2.3.4 --- Expression pattern of foxp4 in adult zebrafish brain --- p.59 / Chapter 3.4 --- Discussion --- p.61 / Chapter Chapter 3 --- Functional analysis of foxp4 in zebrafish embryonic development / Chapter 3.1 --- Introduction --- p.63 / Chapter 3.2 --- Materials and methods --- p.64 / Chapter 3.2.1 --- Materials --- p.64 / Chapter 3.2.2 --- Design of morpholino --- p.68 / Chapter 3.2.3 --- Sequencing of morpholino target regions of foxp4 --- p.70 / Chapter 3.2.4 --- Microinjection --- p.70 / Chapter 3.2.4.1 --- Preparation of materials and equipment --- p.70 / Chapter 3.2.4.2 --- Preparation of injection needle --- p.70 / Chapter 3.2.4.3 --- Preparation of morpholinos --- p.70 / Chapter 3.2.4.4 --- Calibration of injection volume --- p.71 / Chapter 3.2.4.5 --- Microinjection of zebrafish embryo --- p.71 / Chapter 3.2.5 --- Western blotting to assay foxp4 translation inhibition --- p.72 / Chapter 3.2.5.1 --- Preparation of protein extracts --- p.72 / Chapter 3.2.5.2 --- Coomassie blue staining --- p.73 / Chapter 3.2.5.3 --- Western blotting --- p.74 / Chapter 3.2.6 --- Whole mount in-situ hybridization --- p.74 / Chapter 3.3 --- Results --- p.75 / Chapter 3.3.1 --- MO1 knockdown efficiency assayed by Western blotting --- p.75 / Chapter 3.3.2 --- General morphology of morphants --- p.77 / Chapter 3.3.3 --- Histology at the hindbrain region showing the phenotype --- p.79 / Chapter 3.3.4 --- Whole mount in-situ hybridization of different molecular markers --- p.81 / Chapter 3.4 --- Discussion --- p.85 / Chapter Chapter 4 --- Future directions and conclusion / Chapter 4.1 --- Future directions --- p.89 / Chapter 4.2 --- Conclusion --- p.91 / Reference --- p.92

Although much more work needs to be done to elucidate the functional roles of FSH and LH in fish reproduction, the preset study provides a relatively comprehensive study for us to understand the potential roles of FSH and LH during ovarian development in fish, especially the importance of FSH. / At the same time, functional studies were carried out to examine and compare bioactivities of the CHO-derived zfFSH and zfLH in zebrafish ovary, which is the major part of the present project. The following aspects were covered to investigate the actions of zfFSH and zfLH: steroidogenesis and folliculogenesis. / Both recombinant zfGTHs stimulated activin betaA expression but slightly suppressed activin betaB expression. During short-term treatment, zfFSH and zfLH exhibited similar stimulatory effects on activin betaA expression; the effect of zfLH became more prominent after 24 h treatment while zfFSH had little effect. / Previously, our laboratory had established two stable Chinese hamster ovary (CHO) cell lines expressing recombinant zebrafish FSH (zfFSH) and LH (zfLH). However, the production yields are very low. Therefore, the present study tried to adopt the yeast Pichia pastoris as another bioreactor to produce recombinant zfFSH and zfLH. Two different forms of expression vectors for a native form and a fusion form carrying a His-tag, respectively, were constructed for each hormone. Their bioactivities were monitored and confirmed by receptor-based reporter gene assays as well as ovarian fragment incubation. As expected, the native form exhibited much higher activities than the fusion form. / The pituitary gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are the key hormones controlling vertebrate reproduction. Although the two gonadotropins have been characterized in numerous teleost species, our understanding of their biological functions remains rather limited. This is largely due to the lack of pure form of homologous gonadotropins and inadequate understanding of gonadal physiology in most species studied as well as species variation of hormone actions. The present study aims at systematically investigating the functional roles of FSH and LH in the ovary using zebrafish as the model. Zebrafish is becoming more and more popular as the model of reproductive and developmental studies due to several advantages. First, though its body size is small, its ovary is relatively large and available all the year around. Second, zebrafish spawns everyday and its development is fast. Last but not least, its bioinformatics information is tremendous compared to other fish models. / We investigated the effects of zfFSH and zfLH on steroidogenesis by examining the regulation of aromatase by these two hormones. Aromatase catalyzes the conversion of androgens into estrogens during steroidogenesis. Both recombinant zfGTHs stimulated the aromatase expression during short-term treatment (8 h) in ovarian fragment culture, with zfFSH much more potent than zfLH. However, zfFSH continued to exhibit powerful effect on aromatase expression after 24 h treatment while zfLH had little effect at all. The stimulatory effect of zfFSH on aromatase expression was time-, dose- and stage-dependent and was also confirmed by in vivo study. Furthermore, it was also zfFSH but not zfLH that significantly stimulated StAR protein expression during short-term treatment. StAR protein is critical to steroidogenesis by facilitating the movement of cholesterol across the mitochondrial membrane. / zfLH was found to be able to induce GVBD in zebrafish, as demonstrated in other fish species. However, our preliminary data showed that zfFSH was also involved in this process. To our knowledge, this is the first time to demonstrate that homologous FSH induces GVBD in teleosts. / Yu, Xiaobin. / Adviser: Wei Ge. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis submitted in: December 2008. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 152-181). / 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, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Wang Yajun. / "April 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 212-248). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

A critical step in mammalian ovarian follicle development is the transition of gonadotropin-independent preantral follicles to the gonadotropin-dependent antral follicles. However, the molecular mechanisms underlying the transition or early follicle activation are largely unknown. Using zebrafish as the model, we have recently identified Y-box binding protein 1 (YB-1, Ybx1/ybx1), a transcription factor and mRNA binding protein, in early developing oocytes whose expression level was very high in the gonadotropin-independent primary growth (PG) stage but drastically diminished at the beginning of the gonadotropin-dependent secondary growth (SG) stage, i.e., previtellogenic (PV) stage. This has raised interesting questions on the role of Ybx1 in follicle activation as well as how it is controlled. To provide clues to these issues, we first analyzed the regulation of Ybx1 during PG-to-PV transition under IGF-I treatment and the associated signaling pathways. IGF-I, an endocrine/paracrine factor in the growth axis, stimulats Ybx1 phosphorylation via PI3K/Akt but not MAPK pathway in PG follicles. Interestingly, the phosphorylation correlated well with the decline of Ybx1 protein level and the activation of the follicle from the PG follicle pool. This, together with our finding that zebrafish Ybx1 is exclusively produced in PG oocytes in large amount but suddenly disappears during PG-to-PV transition, has prompted us to wonder what the relationship between Ybx1 phosphorylation and degradation. Further experiments showed that Akt directly binds and phosphorylates Ybx1, leading the regulation of Ybx1, including its phosphorylation, cleavage, translocation and degradation, which in turn regulates gene expression and protein synthesis. / In summary, as a multifunctional protein that may play a critical role in early follicle development, Ybx1 is subject to regulation by external factors such as IGF-I, which stimulated Ybx1 phosphorylation via PI3K/Akt but not MAPK pathway. Once Ybx1 is phosphoylated by Akt in the cytoplasm of PG follicle, on one hand, it will be cleaved and translocated to the nucleus to regulate gene expression. On the other hand, the phosphor-Ybx1 can also be degraded through the Ub-proteasome pathway, leading the release of free mRNA to further translation. All these promote the synthesis of many growth- and differentiation-related proteins, which will facilitate early follicle activation. Our findings suggest that the oocyte may serve as the headquarter to programme follicle activation and that the oocyte Ybx1 protein may play a critical role in this event. The delineation of the signaling pathways involved in IGF-I-induced Ybx1 phosphorylation and the regulation of Ybx1 as well as its function in gene transcription and protein synthesis during PG-to-PV transition will provide insight into the mechanism of early follicle activation and puberty initiation. / 哺乳动物卵巢卵泡发育的一个关键步骤是从促性腺激素非依赖的窦前卵泡向促性腺依赖的窦状卵泡的转变过程。但是这一早期卵泡激活的分子机制却不是非常清楚。利用斑马鱼为模型，我们在早期发育的卵母细胞中发现一种名叫Y-box结合蛋白1 （YB-1, Ybx1/ybx1）的转录因子和mRNA 结合蛋白，它在促性腺激素不依赖的初级生长期卵泡（PG）中大量表达，但是在促性腺激素依赖的第二生长期卵泡（SG）,也叫卵黄发生前期(PV)中表达量大大降低。这引发我们猜想YB-1 可能在早期卵泡激活（PG-to-PV 转变）中发挥着重要作用，并且想知道它的这一表达量的巨变是如何被调控的。为了弄清楚这些问题，我们首先分析了IGF-I 处理下Ybx1 在PG-to-PV 的转变中是怎样被调控的，以及相关的信号通路。我们发现在PG 阶段，IGF-I 这种存在于生长轴中的内分泌／旁分泌因子，通过PI3K/Akt 而不是MAPK 通路促进Ybx1 的磷酸化。有趣的是，这种磷酸化的升高正好伴随着Ybx1 蛋白水平的下降以及PG 卵泡的激活。结合我们之前的发现：斑马鱼Ybx1 只在PG 卵母细胞中大量表达但在PG-to-PV 的转变过程中突然消失，促使我们猜想Ybx1 磷酸化和它的降解之间应该存在一定的关系。进一步的实验表明Akt 激酶直接结合并磷酸化Ybx1,导致一系列对Ybx1 调控，包括它的磷酸化，切割，转位以及降解，所有这些又将促进基因的表达调控及蛋白的合成。 / 总之， 多功能蛋白Ybx1 可能在早期卵泡发育过程中发挥着至关重要的作用。外界刺激因子，如IGF-I，通过PI3K/Akt 而非MAPK 途径促进Ybx1 磷酸化。一旦Ybx1 在PG 卵泡细胞质中被Akt 磷酸化，一方面Ybx1 将会被切割并且转位到细胞核中去调节基因表达，另一方面，磷酸化的Ybx1 还会通过泛素蛋白酶途径被降解，从而释放出mRNA 去进一步的翻译。所有这些将促进许多生长和分化相关的蛋白合成，从而促进早期卵泡的激活。我们的研究结果表明，卵母细胞很可能是程序性卵泡激活的核心部分，存在于卵母细胞中的Ybx1 蛋白在这一过程中起着关键作用。研究IGF-I 参与诱导的Ybx1 磷酸化的信号通路以及在PGto-PV 转变过程中对Ybx1 蛋白的调控和它在基因表达及蛋白合成中的作用，将有力的帮助我们弄清早期卵母细胞激活及青春期的启动机制。 / Zhang, Lingling. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 104-127). / Abstracts also in Chinese. / Title from PDF title page (viewed on 06, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Yu, Man Ying Susana. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 103-117). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iv / Acknowledgement --- p.vi / Table of contents --- p.viii / Symbols and abbreviations --- p.xii / Scientific names --- p.xiv / List of figures --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Structure of ovarian follicles --- p.1 / Chapter 1.2 --- Regulation of ovarian follicle development --- p.3 / Chapter 1.2.1 --- Endocrine regulation --- p.3 / Chapter 1.2.1.1 --- Gonadotropins- FSH and LH --- p.3 / Chapter 1.2.1.2 --- Co-gonadotropin- growth hormone --- p.5 / Chapter 1.2.2. --- Paracrine regulation --- p.6 / Chapter 1.2.2.1 --- Activin --- p.6 / Chapter 1.2.2.2 --- Insulin-like growth factor I (IGF-I) --- p.7 / Chapter 1.3 --- The GH-IGF-I axis --- p.7 / Chapter 1.3.1 --- The somatomedin hypothesis --- p.8 / Chapter 1.3.2 --- "Structure and signaling of GH, GHR" --- p.8 / Chapter 1.3.3 --- Structure and signaling of IGF system --- p.9 / Chapter 1.3.4 --- Role of GH-IGF system in reproduction --- p.11 / Chapter 1.3.5 --- GH action in ovarian functions --- p.12 / Chapter 1.3.6 --- IGF-I action in ovarian functions --- p.13 / Chapter 1.3.7 --- The mini GH-IGF axis within the ovary --- p.14 / Chapter 1.4 --- Objectives of present study --- p.14 / Chapter Chapter 2 --- "Expression Profiles of the GH-IGF System in the Ovary of Zebrafish, Danio rerio" --- p.19 / Chapter 2.1 --- Introduction --- p.19 / Chapter 2.2 --- Material and Methods --- p.21 / Chapter 2.2.1 --- Animals --- p.21 / Chapter 2.2.2 --- Isolation of tissues and different stages of follicles from the zebrafish --- p.22 / Chapter 2.2.3 --- Separation of somatic follicle layers and oocytes --- p.22 / Chapter 2.2.4 --- Primary follicle cell culture --- p.22 / Chapter 2.2.5 --- Total RNA extraction --- p.23 / Chapter 2.2.6 --- Reverse transcription --- p.23 / Chapter 2.2.7 --- "Validation of semi-quantitative RT-PCR assays for GH (gh), GHR (ghr), IGF-I (igf1), IGF-II (igf2), and IGF-I receptor (igf1r)" --- p.24 / Chapter 2.2.8 --- Data analysis --- p.25 / Chapter 2.3 --- Results --- p.25 / Chapter 2.3.1 --- Validation of semi-quantitative RT-PCR assays --- p.25 / Chapter 2.3.2 --- Spatial expression of GH-IGF in different tissues of zebrafish --- p.26 / Chapter 2.3.3 --- "Localization of gh, ghr, igf1, igf2 and igf1r within the zebrafish follicle" --- p.26 / Chapter 2.3.4 --- Temporal expression profiles of GH-IGF system during folliculogenesis --- p.28 / Chapter 2.4 --- Discussion --- p.28 / Chapter Chapter 3 --- Regulation of the GH-IGF-I System and Its Cross-talk with the Activin System in the Zebrafish Ovary --- p.43 / Chapter 3.1 --- Introduction --- p.43 / Chapter 3.2 --- Material and methods --- p.45 / Chapter 3.2.1 --- Animals --- p.45 / Chapter 3.2.2 --- Chemicals and hormones --- p.45 / Chapter 3.2.3 --- Primary follicle cell culture --- p.45 / Chapter 3.2.4 --- Preparation of ovarian fragments --- p.45 / Chapter 3.2.5 --- Total RNA extraction --- p.45 / Chapter 3.2.6 --- RT-PCR --- p.47 / Chapter 3.2.7 --- Construction of real-time PCR standards --- p.47 / Chapter 3.2.8 --- Real-time PCR and semi-quantitative RT-PCR --- p.48 / Chapter 3.2.9 --- Data analysis --- p.49 / Chapter 3.3 --- Results --- p.49 / Chapter 3.3.1 --- "Expression of growth hormone (gh), growth hormone receptors (ghr1 and ghr2\ IGF-I (igf1), IGF-II (igf2), IGF-I receptor (igf1ra and igf1rb), activin subunits (inhba and inhbb) and follistatin (fst) in cultured zebrafish ovarian fragments" --- p.49 / Chapter 3.3.2 --- "Establishment of real-time RT-PCR for zebrafish inhba, inhbb and bactin" --- p.50 / Chapter 3.3.3 --- GH regulation of activin expression in cultured zebrafish follicle cells --- p.50 / Chapter 3.3.4 --- GH regulation of IGF-I in cultured zebrafish follicle cells --- p.51 / Chapter 3.3.5 --- IGF-I regulation of activin expression in cultured zebrafish follicle cells --- p.51 / Chapter 3.3.6 --- Activin regulation of IGF system --- p.52 / Chapter 3.4 --- Discussion --- p.52 / Chapter Chapter 4 --- Production of recombinant zebrafish growth hormone --- p.69 / Chapter 4.1 --- Introduction --- p.69 / Chapter 4.2 --- Material and Methods --- p.71 / Chapter 4.2.1 --- Animals --- p.71 / Chapter 4.2.2 --- Construction of expression plasmids pPIC9K/zfGH --- p.71 / Chapter 4.2.3 --- Production of recombinant zebrafish GH using Pichia pastoris --- p.73 / Chapter 4.2.4 --- SDS-PAGE and silver staining --- p.74 / Chapter 4.2.5 --- Purification --- p.74 / Chapter 4.2.6 --- Primary follicle cell culture --- p.75 / Chapter 4.2.7 --- Zebrafish hepatic cell culture --- p.76 / Chapter 4.2.8 --- RNA extraction and RT-PCR --- p.76 / Chapter 4.2.9 --- Real-time PCR --- p.77 / Chapter 4.2.10 --- Cell culture and transient transfection --- p.78 / Chapter 4.2.11 --- Luciferase reporter gene assay --- p.78 / Chapter 4.2.12 --- Data analysis --- p.79 / Chapter 4.3 --- Results --- p.79 / Chapter 4.3.1 --- Production of recombinant zebrafish GH --- p.79 / Chapter 4.3.2 --- Effect of recombiant zfGH on the expression of activin β Aand βB in cultured zebrafish follicle cells --- p.80 / Chapter 4.3.3 --- Effect of zfGH on the expression of igf1 in cultured zebrafish hepatic cells --- p.80 / Chapter 4.3.4 --- Luciferase reporter gene assay --- p.81 / Chapter 4.4 --- Discussion --- p.81 / Chapter Chapter 5 --- General Discussion --- p.94 / Chapter 5.1 --- Overview --- p.94 / Chapter 5.2 --- Major achievements of the present study --- p.95 / Chapter 5.2.1 --- Demonstration of a local mini-GH-IGF-I axis within the zebrafish ovary --- p.96 / Chapter 5.2.2 --- Differential expression profiles of the GH-IGF system during folliculogenesis --- p.96 / Chapter 5.2.3 --- The inter-relationship of GH-IGF and activin-follistatin systems --- p.96 / Chapter 5.2.4 --- Production of recombinant zebrafish GH --- p.97 / Chapter 5.3 --- Future prospects --- p.97 / References --- p.102 / Symbols and Abbreviations / Symbols / α Alpha / β Beta / Abbreviations / 20β-HSD 20β-hydroxysteroid dehydrogenase / bp Base pair / cAMP Cyclic adenosine monophosphate / cDNA Complementary cDNA / CHO Chinese hamster ovary / "DHP 17α, 20β-dihydroxy-4-prenane-3 -one" / DNA Deoxyribonucleic acid / EGF Epidermal growth factor

I am interested in discovering the role of field potential oscillations in producing synchrony within the song system of the male zebra finch brain. An important function attributed to neural synchrony is sensorimotor integration. In the production of birdsong, sensorimotor integration is crucial, as auditory feedback is necessary for the maintenance of the song. A cortical-thalamic-cortical feedback loop is thought to play a role in the integration of auditory and motor information for the purpose of producing song. Synchronous activity has been observed between at least two nuclei in this feedback loop, MMAN and HVC. Since low frequency field potential oscillations have been shown to play a role in the synchronization of nuclei within the brain of other model animals, I hypothesized that this may be the case in the zebra finch song system. In order to investigate whether oscillatory activity is a mechanism behind the synchronous activity observed between HVC and MMAN, I performed dual extracellular recordings of neural activity within the zebra finch song system. Results suggest that oscillations are likely not involved in the synchrony observed in these nuclei. Future study may reveal that the structure of the feedback loop is necessary, and possibly even sufficient, for the synchronous activity in the zebra finch song system.

Thesis (M.S.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 35-40). Also available on the World Wide Web.