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

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

University of Macau / Institute of Chinese Medical Sciences

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 234-254). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 234-254). Also available on the Internet.

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 25, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. Anand Chandrasekhar. Vita. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2009. / Title from first page of PDF file. Includes bibliographical references (p. 153-165).

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 187-198). Also available in electronic version. Access restricted to campus users.

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