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A nonrecirculatory liver slice perifusion system has been developed and utilized for investigating glucagon-stimulated glycogenolysis in normal and diabetic states. It has been shown here that slices maintained in this system experience a controlled environment with respect to temperature and pH and remain viable throughout a three-hour experimental period based upon their maintenance of intracellular potassium levels. Although glycogen content falls by 40%, slices exhibit significant glycogenolysis in a dose-response manner upon challenge with glucagon, with maximal concentrations eliciting a 2.2-fold stimulation. This system, which permits nonrecirculatory challenge of liver tissue and subsequent analysis of both intracellular events and overall physiological responses, is extremely useful for examining hormonal mechanisms operating for glucagon, particularly at low concentrations. Using this methodology, liver slices challenged with glucagon exhibit a biphasic dose-response for glycogenolysis. While the second phase parallels cAMP (cyclic adenosine 3':5'-monophosphate) accumulation and cAMP-PK (cAMP-dependent protein kinase) activation, the first is mediated independent of cAMP. Trinitrophenylhistidine-1, homoarginine-12-glucagon (THG), which can antagonize glucagon-stimulated adenylate cyclase, exhibits 50% partial agonist activity for cAMP production and cAMP-PK but full agonism for glycogenolysis. Separation between these events is only two-fold indicating a cAMP-mediated process. [Des-amino-fYRKKE]-glucagon, ([Des-amino-His¹,D-Phe⁴,Tyr⁵,Arg¹²,Lys¹⁷·¹⁸,Glu²¹]-glucagon), another adenylate cyclase antagonist, does not stimulate cAMP or cAMP-PK up to 25 μM yet still elicits glycogenolysis. These results demonstrate that glucagon does indeed stimulate both cAMP-independent as well as cAMP-dependent glycogenolysis in normal liver. In diabetic systems, glucagon elicits attenuated adenylate cyclase activity in liver plasma membranes with reduction in basal activity and extent of stimulation. Maximal stimulation of cAMP production is also reduced by half in liver slices, but in both systems (normal vs. diabetic) EC₅₀ values for cAMP production are identical. Neither THG nor [des-amino-fYRKKE]-glucagon stimulate cAMP production or cAMP-PK in diabetic liver slices. While THG lowers blood glucose levels in vivo, [des-amino-fYRKKE]-glucagon acts as an agonist. These results suggest that the mechanisms which operate for glucagon-stimulated glycogenolysis in normal liver are attenuated in the diabetic state. Furthermore, antagonism of cAMP production alone is insufficient to antagonize glucagon's overall physiological action.

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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

In this study, both physiological and cellular effects are elicited by natriuretic peptides (NPs), a novel type of plant hormone. It was found that rat ANP (rANP) influenced stomatal opening movement in Tradescantia sp., where a significant increase in stomatal opening was observed in the presence of 1 µM rANP. Furthermore, this effect is mediated by cGMP, a (putative) second messenger of NPs. Two inhibitors of guanylyl cyclase, LY 83583 and methylene blue, inhibited rANP-induced stomatal opening. In contrast, stomatal opening is induced in a concentration dependent manner by the cell permeant cGMP analogue 8-Br-cGMP. In addition it was found, that like in animals, the secondary structure of rANP is essential for rANP responses. Linearised rANP is biologically inactive. Since ANP elicit plant responses, an attempt was made to isolate NP analogues from plants. A protocol for partially purifying NP from plants was developed. It was found that two fractions eluted from an immunoaffinity chromatography column (0.5 M KCI eluted fraction and 0.75 M KCI eluted fraction) were biologically active. The level of cGMP in response to NPs was also tested. It is suggested that the receptor of NP is specific since only 0.75 M KCI eluted fractions increased cGMP levels in Zea mays root stele tissue. rANP did not elicit an effect on cGMP levels in this tissue and LY 83583 did not affect this response. It is therefore argued that a plant specific biologically active NP system is present in the stele and it is predicted that NPs modulate solute movement in this tissue. NPs also influence K⁺, Na⁺ and H⁺ fluxes in Zea mays root stele. Increase in both K⁺ and Na+ uptake were observed after 30 min., while H⁺ flux shifted immediately toward influx in the presence of both 0.5 and 0.75 KCI eluted fractions. Finally, a model is proposed for the effect of NPs on solute movement and its signalling system in plants.

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