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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

A study of microRNA-132 and -212 in murine granulosa cells during folliculogenesis

Lin, Sau-wah, Selma., 林秀華. January 2010 (has links)
published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy
2

GHRH/PACAP-GH-IGF axis in the ovary of zebrafish, Danio rerio. / CUHK electronic theses & dissertations collection

January 2012 (has links)
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
3

Growth and differentiation factor 9 (GDF9) in the ovary of zebrafish, danio rerio. / CUHK electronic theses & dissertations collection

January 2006 (has links)
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.
4

Studies on the molecular regulation of ovarian maturation in penaeid shrimp. / CUHK electronic theses & dissertations collection

January 2008 (has links)
Another important gene, heat shock factor (MeHSF) was also cloned using homology based PCR because it was suggested to participate in the transcriptional regulation of many essential components of ovarian maturation including vitellogenin gene and several proteins for hormones metabolism. The complete cDNA sequence of MeHSF was 2211 bp in length, which encoded a 622 amino acid protein. The translated MeHSF protein shared high similarity with those of other species, especially in the N terminal region. RT-PCR showed that MeHSF was universally expressed in most of the female tissues investigated including ovary, central nervous system, heart, gill, gut and muscle. However, its expression was not detectable in eyestalk and hepatopancreas. MeHSF was highly expressed in immature ovaries, and decreased dramatically with the progress of ovarian maturation. Since the synthesis of vitellogenin in ovary showed an opposite trend, the result suggested that MeHSF probably functioned as a transcriptional repressor to vitellogenin. Four HSFs isoforms generated from alternative splicing were obtained in immature ovaries, suggesting a possible universal role of HSF in coordinating transcription of different target genes during shrimp ovarian maturation. / As an important component of enzymatic scavenger systems, glutathione peroxidases (GPx) play important roles in maintaining the balance between reactive oxygen species (ROS) production and cellular scavenging ability. In this research, a full length GPx gene (MeGPx) which had been identified using RAP-PCR previously was cloned and characterized. MeGPx might play a pivotal role in preventing oocytes from oxidative damage and balancing ROS production. The present data on shrimp GPx provides insights on the regulation of ROS in the ovarian maturation process. / Four candidate genes possibly participating in the regulation of ovarian maturation were obtained by random sequencing the libraries, including metallothionein, two zinc finger proteins and member 4 of wingless-type MMTV integration site family (WNT4). The zinc finger protein containing a plant homeodomain, was only expressed in the eyestalk of female with immature ovaries, but not that of female with early mature and mature ovaries. The full length cDNA sequence of shrimp WNT4 gene (MeWNT4) was obtained using RACE technique. RT-PCR showed that the expression of MeWNT4 in eyestalk decreased with the maturation of shrimp ovaries. Interestingly, MeWNT4 was strongly expressed in the central nervous system and gut of both female and male shrimp. It was suggested that WNT4 could antagonize the testis determining factor (SRY), and play an essential role in suppressing the formation of testis, and at the same time, controlling of female development. Thus, the identification WNT4 from crustacean would contribute to our understanding on the sex determination mechanism. / In this thesis, two genes, heat shock protein 90 and heat shock factor, possibly playing important roles in shrimp ovarian maturation were identified and characterized. / Shrimp farming plays an important economic role in Southeast Asian countries. Yet further development of this industry is seriously restricted by the environmental deterioration and the prevalence of fetal diseases. Moreover, the failure of sexual maturation of cultured female shrimp forms a bottleneck to the further development of shrimp aquaculture. With the aim to produce shrimp without totally depending on the wild stocks, many studies have been focused on the endocrine regulation of shrimp ovarian maturation. In order to enhance our understanding on the molecular events occurred during ovarian maturation, in this research, several candidate genes are identified, and their potential roles in ovarian maturation are studied in the shrimp Metapenaeus ensis. / Since heat shock protein 90 gene is one of the essential components of steroid hormone signal cascades in vertebrates, it was cloned and isolated by homology cloning strategy. The complete cDNA sequence of shrimp Hsp90 ( MeHsp90) was 2524 by in length, which encoded a 720 amino acid polypeptide. The MeHsp90 coding region was interrupted by four introns. MeHsp90 was differentially expressed in eyestalk, ovary and hepatopancreas at different ovarian maturation stages, and consistently expressed in other tissues including heart, gill, gut, muscle and central nervous system. In vitro ovary explant assay revealed that MeHsp90 expression in immature ovary could be induced by the addition of exogenous estradio1-17beta. MeHsp90 was highly expressed in pre-vitellogenic oocytes, and its expression decreased with the progress of maturation, and finally stopped in late-vitellogenic oocytes. The co-regulation of MeHsp90 and vitellogenin by estrogen hormone suggested a possible regulatory role of Hsp90 in vitellogenin synthesis of the shrimp. / Wu, Long Tao. / "May 2008." / Adviser: Chu Ka Hou. / Source: Dissertation Abstracts International, Volume: 70-03, Section: B, page: 1409. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (p. 92-113). / 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.
5

A potential mechanism for follicle activation in zebrafish: the role of IGF-I/Ybx1 in the primary growth follicle of zebrafish / CUHK electronic theses & dissertations collection

January 2015 (has links)
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.
6

Expression control of zebrafish gonadotropin receptors in the ovary. / CUHK electronic theses & dissertations collection

January 2012 (has links)
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

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