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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328140 |
| Date | January 2012 |
| Contributors | Chen, Weiting, Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xiii, 120 leaves) : ill. (chiefly col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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