Thesis (Ph.D.)--University of Melbourne, Dept. of Zoology, 2004. / Typescript (photocopy). Includes bibliographical references (leaves 202-234).

Thesis (M.S.)--Springfield College, 2004. / Includes bibliographical references. Also available online (PDF file) by a subscription to the set or by purchasing the individual file.

Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Thesis (M.S.)--Springfield College, 2004. / Includes bibliographical references.

No description available.

Recent studies have shown that postmenopausal follicle stimulating hormone (FSH) levels may be predictive of future cardiovascular disease risk. However, little is known about postmenopausal FSH levels, including the level of variation between women and factors associated with this variation. We assessed the relationship of multiple reproductive factors with FSH levels among 588 postmenopausal women in the Kuopio Ischemic Heart Disease Risk Factor Study. Participants were aged 53 to 73 years and not using hormone therapy at baseline (1998-2001). Reproductive factors were assessed at baseline, along with FSH levels. After adjustment for sex steroids, adiposity measures, plasma lipids, blood pressure, and behavioral factors, we observed that women with 3 or more births and an age at first birth of 25 or later had mean FSH levels that were significantly 7.6 IU/L lower than those of women with a 1 to 2 births and an age at first birth of 24 or less. Number of miscarriages was inversely correlated with FSH levels. Women reporting a 7 or more years of OC use and 4-6 or 7 or more years of HT use each had significantly higher mean FSH levels than women who had never used OCs or HT. In summary, multiple reproductive factors were associated with postmenopausal FSH levels, independent of estradiol, adiposity, and other factors. These findings warrant replication and further exploration of potential underlying mechanism.

Ko Nga Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 98-131). / Abstracts in English and Chinese. / Abstract (in English) --- p.ii / Abstract (in Chinese) --- p.v / Acknowledgements --- p.vii / Table of Contents --- p.ix / List of Figures --- p.xiv / List of Tables --- p.xvii / Symbols and Abbreviations --- p.xviii / Scientific Names --- p.xxi / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Gonadotropins --- p.1 / Chapter 1.1.1 --- Structure --- p.1 / Chapter 1.1.2 --- Function --- p.3 / Chapter 1.1.3 --- Regulation --- p.5 / Chapter 1.1.3.1 --- Hypothalamic regulators (GnRH) --- p.5 / Chapter 1.1.3.2 --- Endocrine regulators from gonads (steroids) --- p.7 / Chapter 1.1.3.3 --- Paracrine regulators (activin) --- p.9 / Chapter 1.1.4 --- Promoter analysis --- p.9 / Chapter 1.2 --- Activin Family of Growth Factors --- p.12 / Chapter 1.2.1 --- Activin --- p.12 / Chapter 1.2.1.1 --- Structure --- p.12 / Chapter 1.2.1.2 --- Function --- p.13 / Chapter 1.2.1.3 --- Signaling --- p.15 / Chapter 1.2.2 --- Follistatin --- p.16 / Chapter 1.2.2.1 --- Structure --- p.16 / Chapter 1.2.2.2 --- Function --- p.17 / Chapter 1.3 --- Objectives --- p.18 / Chapter Chapter 2 --- Establishment and Characterization of Stable LβT2 Cell Lines Containing and Expressing SEAP Driven by the Goldfish FSHβ Promoter / Chapter 2.1 --- Introduction --- p.29 / Chapter 2.2 --- Materials and Methods --- p.31 / Chapter 2.2.1 --- Construction of expression plasmid --- p.31 / Chapter 2.2.2 --- Cell culture --- p.32 / Chapter 2.2.3 --- Cotransfection of LβT2 cells --- p.32 / Chapter 2.2.4 --- G418 selection of transfected LpT2 cells --- p.33 / Chapter 2.2.5 --- SEAP reporter gene assay --- p.33 / Chapter 2.2.6 --- Cloning of pSEAP/gfFSHβ promoter and pBK- CMV-transfected LβT2 cells by limited dilution --- p.34 / Chapter 2.2.7 --- Extraction of genomic DNA --- p.34 / Chapter 2.2.8 --- Isolation of total RNA --- p.35 / Chapter 2.2.9 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.35 / Chapter 2.3 --- Results --- p.36 / Chapter 2.3.1 --- Optimization of G418 concentration for selection --- p.36 / Chapter 2.3.2 --- Expression of SEAP reporter gene by pSEAP/gfFSHβ promoter and pBK-CMV-transfected LβT2 cells --- p.37 / Chapter 2.3.3 --- Establishment of LβT2 cell lines that contain a functional gfFSHp promoter --- p.37 / Chapter 2.3.4 --- Characterization of LβT2#23 that contains a functional gfFSHβ promoter --- p.38 / Chapter 2.4 --- Discussion --- p.39 / Chapter Chapter 3 --- Hormonal Regulation of Goldfish Follicle-Stimulating Hormone β (FSHβ) Promoter Activity in LpT2#23 Cells / Chapter 3.1 --- Introduction --- p.52 / Chapter 3.2 --- Materials and Methods --- p.54 / Chapter 3.2.1 --- Cell culture --- p.55 / Chapter 3.2.2 --- Drug treatment --- p.56 / Chapter 3.2.3 --- SEAP reporter gene assay --- p.56 / Chapter 3.2.4 --- Isolation of total RNA --- p.57 / Chapter 3.2.5 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.57 / Chapter 3.2.6 --- Data analysis --- p.58 / Chapter 3.3 --- Results --- p.59 / Chapter 3.3.1 --- Effects of goldfish activin on FSHβ promoter --- p.59 / Chapter 3.3.2 --- Blockade of activin effects by follistatin --- p.59 / Chapter 3.3.3 --- Effects of different hormones and steroids on FSHβ promoter --- p.60 / Chapter 3.4 --- Discussion --- p.61 / Chapter Chapter 4 --- Promoter Analysis for the Activin Responsive Element (ARE) in the Goldfish Follicle-Stimulating Hormone β (FSHβ) Gene / Chapter 4.1 --- Introduction --- p.71 / Chapter 4.2 --- Materials and Methods --- p.74 / Chapter 4.2.1 --- Generation of SEAP reporter plasmids containing the gfFSHβ promoter of different lengths --- p.74 / Chapter 4.2.2 --- PCR screening and restriction analysis --- p.75 / Chapter 4.2.3 --- Midiprep --- p.76 / Chapter 4.2.4 --- Cell culture --- p.77 / Chapter 4.2.5 --- Transfection of the pSEAP/gfFSHβ promoter constructs into LβT2 cells --- p.77 / Chapter 4.2.6 --- Activin treatment --- p.77 / Chapter 4.2.7 --- SEAP assay --- p.78 / Chapter 4.3 --- Results --- p.78 / Chapter 4.3.1 --- Subcloning of the gfFSHβ promoter of decreasing length into SEAP reporter vector --- p.78 / Chapter 4.3.2 --- Activin stimulation of the pSEAP/gfFSHβ promoter constucts in LβT2 cells --- p.79 / Chapter 4.4 --- Discussion --- p.80 / Chapter Chapter 5 --- General Discussion / Chapter 5.1 --- Overview --- p.92 / Chapter 5.2 --- Contribution of the present research --- p.95 / Chapter 5.2.1 --- Establishment of stable LβT2 cell lines containing and expressing SEAP driven by gfFSHβ promoter --- p.95 / Chapter 5.2.2 --- Hormonal regulation of the gfFSHβ promoterin LβT2#23 cells --- p.95 / Chapter 5.2.3 --- Identification of the activin responsive element (ARE) on the gfFSHβ promoter --- p.96 / Chapter 5.3 --- Future research direction --- p.96 / References --- p.98

Master of Science / Department of Animal Sciences and Industry / Timothy G. Rozell / Timothy G. Rozell / An increase in pre-mating dietary energy positively influences ovulation and lambing rates, and this practice is known as nutritional flushing. The mechanisms of flushing, however, are still unknown. Increasing dietary energy approximately two weeks before breeding likely increases the production of insulin-like growth factor I (IGF-I) within the ovary, which stimulates the synthesis of follicle stimulating hormone receptor (FSHR). Several alternatively spliced transcripts of the FSHR have been identified in sheep. Each variant form is believed to be produced according to the stage of follicle development. This study was carried out to evaluate expression patterns of the FSHR variant forms (FSHR-1, FSHR-2 and FSHR-3) in the sheep ovary in response to different flushing diets. For this experiment, yearling Rambouillet ewes (n=93) were allocated among 6 different energy type treatment diets, either prairie or alfalfa hay based, for at least two weeks in combination with the insertion of a controlled internal drug releasing device (CIDR). Two of the treatment groups had commercially available block supplements provided and two had rolled corn supplemented. Mid-ventral laparotomy was performed on each ewe 3.5 to 4 days after CIDR removal. Follicles 4 mm and greater were aspirated and categorized as either medium (M; 4 to 6 mm) or large (L; > 6 mm). Total RNA was extracted from granulosa cells (GC) and reverse transcribed followed by qPCR of the resulting cDNA using specifically designed primer sets for each variant of the FSHR and for the LH receptor. Changes in live weight were different (P < 0.01) between treatment diets but there were no statistical differences for NEFA concentrations between any of the treatments nor were there differences for body condition (mean = 3.0) or lambing rate. Therefore, it is likely a flushing response did not occur in this study. Expression of FSHR-1was different between M and L follicles (P < 0.01) and tended to be different for ewes fed alfalfa hay (P = 0.05). Overall mean expression of FSHR-3 was greater than expression of FSHR-1 or FSHR-2 (P < 0.01), although there was no difference between M and L follicles, or between treatment diets. The concentration of estradiol in follicular fluid was not different between the treatment diets or follicle sizes nor was expression of lutenizing hormone receptor (LHR), indicating that follicles were similar developmentally. The FSHR-1 form seemed to be the variant most likely to be involved in later stages of follicular development, and is potentially involved in follicle rescue. For all follicles, FSHR-3 was the more highly expressed form of the FSHR and may likely be essential throughout antral follicle development. Further research is required to determine the exact mechanism whereby initial energy status of ewes seems critical for the increased ovulation rate that occurs after energy supplementation (i.e. the flushing response).

Orientador: Ciro Moraes Barros / Banca: Ronaldo Luiz Ereno / Banca: João Carlos Ferreira / Resumo: O crescimento folicular em bovinos ocorre em um padrão de ondas que pode variar de 2 a 3 por ciclo estral e caracteriza-se pelo crescimento sincrônico de um grupo de folículos antrais, onde apenas um se tornará dominante. A quantidade de folículos recrutados é variável entre os animais, porém apresenta alta repetibilidade entre os indivíduos. Animais Bos indicus apresentam maior quantidade de folículos recrutados por onda que animais Bos taurus. Objetivou-se com o presente trabalho, (1) identificar fêmeas das raças Nelore (Bos indicus) e Aberdeen Angus (Bos taurus), que possuam elevado ou reduzido número de folículos recrutados por onda e relacionar o número de folículos à concentração plasmática do hormônio anti-mulleriano (AMH); (2) relacionar o numero de folículos antrais à fertilidade de fêmeas Nelore e; (3) determinar se o tratamento superestimulatório altera as concentrações plasmáticas de AMH em vacas Nelore. No experimento 1, contagem do numero de folículos antrais das fêmeas Nelore (n=20) e Angus (n=30) foi feita por duas avaliações ultrassonográficas (US). Considerando a média  EPM da contagem de folículos antrais de cada raça, as novilhas foram classificas em alta contagem folicular (ACF; n= 9 novilhas Nelore e 15 Angus) ou baixa contagem folicular (BCF; n= 11 novilhas Nelore e 15 Angus). O AMH foi mensurado por Elisa em uma amostra de sangue coletada durante a ultima US. No experimento 2, foram utilizadas novilhas da raça Nelore (n=86) submetidas a um protocolo de inseminação artificial em tempo fixo (IATF). As novilhas foram classificadas (avaliação US no D0 da IATF) em 3 grupos: ACF (>40 folículos; n=29), intermediário (ICF;>20 e <40 folículos; n=34) e BCF (<20 folículos; n=23). No experimento 3, foram utilizadas 20 vacas da raça Nelore de ICF e ACF. Os animais foram separados pela contagem folicular e distribuídos em 2 grupos: Grupo controle IATF (n=9) e grupo P36 (superestimuladas ... / Abstract: In bovines follicular development happens in a wave pattern that can vary from two to three waves per estral cycle with a group of follicles growing simultaneously but with only ano becoming the dominat follicle. The number of recruited follicle is highly variable between animal; however it is highly repeatable within an animal. Bos indicus animals present greater number of follicles per wave than Bos taurus animals. The objective of the present study was 1) identify animals from the Nelore and Abeerden Angus breed that have high or low number of follicles recruited per follicle wave and relate the number of follicles with AMH concentration; 2) to relate the number of antral follicles with the fertility of Nelore females; 3) to determine if the superstimulatory treatment alters the plasmatic concentrations of AMH. In experiment 1 the follicle count in Nelore and Angus heifers was done through two ultrasound examinations (US). Considering the mean + SEM of the follicle count in each breed, the heifers were grouped in high (HFC) or low (LFC) follicle count. Plasmatic AMH was measured in a blood smple drawn at the last US. In Experiment 2, there were used Nelore heifers submitted to a TFAI protocol. Heifers were grouped in HFC (>40 follicles), intermediary (>20 an <40 follicle) and LFC (<20 follicle). In experiment 3, Nelore cows with high and intermediary follicles counts were used. Animals were separated in control TFAI group and P36 group. Blood samples for AMH measurement were collected on day 5 (P36) and on day 8 (TFAI and P36). Result from experiment 1 were analyzed using ANOVA. Experiment 2 was analyzed using a logistic regression and experiment 3 was ... / Mestre

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