CUHK electronic theses & dissertations collection

卵巢是女性生殖系統中一個重要的器官，負責為受精提供卵子，以及合成生殖過程中所必需，同時也在其他生理過程中起重要作用的各種激素。大約有30%的不育源於卵巢的問題，包括無排卵，無月經，月經週期不規律和激素水平異常等。雄激素:雌激素比例過高，卵泡發育異常，無排卵等卵巢功能障礙常見於各種疾病中，例如多囊性卵巢綜合征(PCOS)--一種影響5~10%育齡婦女的內分泌疾病，以及囊性纖維化( CF)--一種由囊性纖維化跨膜電導調節器(CFTR) 基因突變引起的遺傳疾病。然而引起這些卵巢功能障礙的確切機制並不清楚。雌激素是在卵泡雌激素(FSH) 的調節下，在卵巢顆粒細胞中通過芳香化臨的住激素轉化而生成的。在論文第一部分的研究中，我們旨在證明CFTR 在卵巢顆粒細胞中的表達，以及它參與雌激素生成的過程。實驗結果證實了CFTR 在小鼠和人顆粒細胞中的表達，同時表明CFTR 通過一種碳酸氫根離子(HC0₃⁻) 敏感的可溶性腺苦酸環化梅(sAC) ，放大FSH 所刺激的雌激素生成過程。實驗結果顯示，在原代小鼠顆粒細胞中， HC0₃⁻能夠增強FSH 所引起的CREB 磷酸化，芳香化晦表達，以及雌激素的生成，而在抑制CFTR 的情況下，或在CFTR 敲除/DeltaF508 突變小鼠的顆粒細胞中， HCO3-的放大作用顯著降低。CFTR 和芳香化醋的表達水準在人顆粒細胞中具有正相關性，進一步支持CFTR 對雌激素生成的調節作用。在PCOS 患者的顆粒細胞和大鼠PCOS 模型的卵巢中， CFTR 和芳香化醋的表達水準顯著下調。這些結果提示， CFTR 對雌激素生成調節這一機制的缺陷可能參與了CF 和PCOS 中卵巢功能障礙的發病機理。卵泡發育很大程度上依賴於顆粒細胞的增殖'生存和凋亡，這些過程在PCOS 中都會出現異常。論文的第二部分冒在研究顆粒細胞的CFTR 在PCOS 的卵泡發育異常中的作用。實驗結果表明， CFTR 在PCOS 大鼠的囊，性卵泡的顆粒細胞中表達降低，同時伴隨著PCNA 和Bcl-2 的下調，而Bax 和cleaved caspase-3則沒有變化，提示顆粒細胞的增殖和生存/抗凋亡能力降低。敲減或抑制顆粒細胞中的CFTR 導致細胞存活降低， PCNA 和Bcl-2 表達下調，以及細胞凋亡增加，提示CFTR 對顆粒細胞增殖和生存的調節作用。CFTR 通過HC0₃⁻/ sAC/PKA 信號通路，調節基礎及FSH 刺激引起的ERK I!2 磷酸化，及其下游的CyclinD2 和PCNA表達，從而促進顆樹圍胞的增殖。顆粒細胞CFTR 的下調可能通過抑制細胞增殖和降低細胞生存能力，參與了PCOS 中的囊性卵泡的形成過程。綜上所述，本論文證明了CFTR 在卵巢顆粒細胞上的表達，並且參與調節顆粒細胞雌激素生成和細胞的增殖和生存。CFTR 的缺陷或表達下調可能是導致CF和PCOS 的卵巢功能障礙的發病機理。The ovary is the female reproductive organ, which produces female gametes, oocytes for fertilization and sex hormones essential to reproduction and important to a wide range of physiological and pathological events as well. About 30% of infertility cases arise from ovarian problems, including anovulation, amenorrhea, irregular menstrual cycle and abnormal hormone levels. Ovarian disorders, such as high androgen to estrogen ratio, abnormal folliculogenesis and anovulation, are often seen in diseases, including polycystic ovarian syndrome (PCOS) and cystic fibrosis (CF). The former is an endocrine disorder affecting 5~10% women of reproductive age, and the latter is a common genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR). However, the exact mechanisms underlying the ovarian disorders seen in these diseases are not well understood.Estrogen biosynthesis is profoundly influenced by follicle-stimulating hormone (FSH) that regulates the conversion of androgen to estrogen in ovarian granulosa cells by the rate-limiting enzyme aromatase. The first part of the study aims to investigate the expression of CFTR in granulosa cells and its involvement in regulating estrogen production. The results demonstrate the expression of CFTR in both mouse and human granulosa cells, and provide evidence demonstrating a previously unsuspected role of CFTR in amplification of FSH-stimulated ovarian estrogen biosynthesis and the involvement of a HC0₃⁻ sensor, the soluble adenylyl cyclase (sAC) in this synthesis. FSH-stimulated CREB phosphorylation, aromatae expression, as well as estradiol production are enhanced by HC0₃⁻ and sAC, which could be significantly reduced by CFTR inhibition or in ovaries or granulosa cells of cftr knockout/deltaF508 mutant mice. The fact that CFTR expression is found positively correlated with aromatase expression in human granulosa cells supports its role in regulating estrogen production in humans. Reduced CFTR and aromatase expression is also found in polycystic ovarian syndrome (PCOS) rodent models and human patients. These findings suggest that defective CFTR-dependent regulation of estrogen production may underline the ovarian disorders seen in CF and PCOS.Folliculogenesis largely depends on the proliferation, survival and apoptosis of granulosa cells in the follicles and alteration in which has been found in PCOS. The second part of the study aims to investigate the possible involvement of granulosa cell CFTR in the impaired folliculogenesis in PCOS. The results show that downregulation of CFTR is found in the cystic follicles, which is accompanied by reduced expression of PCNA and Bcl-2, but not Bax and cleaved caspase-3, in the ovaries of PCOS rat models, indicating reduced cell proliferation and survival/anti-apoptotic ability. Knockdown or inhibition of CFTR in granulosa cell culture results in reduced cell viability, downregulation of PCNA and Bcl-2 and increase of apoptosis, supporting a role of CFTR in regulating granulosa cell proliferation and survival. CFTR exerts its effect on granuloa cell proliferation by modulating basal and FSH-stimulated ERKl/2 phosphorylation and the expression of its downstream target CyclinD2 and PCNA through the HC0₃⁻/sAC/PKA pathway. These findings suggest that downregulation of CFTR may play a role in the formation of cystic follicles by inhibiting granulosa cell proliferation and reducing cell survival ability, therefore providing a possible mechanism for the abnormal folliculogenesis in PCOS.In conclusion, the present study has demonstrated the expression of CFTR in the ovarian granulosa cell and its role in regulation of granulosa cell proliferation, survival and estrogen production. Defect of CFTR in CF and downregulation of CFTR in PCOS may contribute to the abnonnal honnone profile and impaired folliculogenesis in both disease conditions.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Chen, Hui."October 2011."Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.Includes bibliographical references (leaves 124-137).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 --- p.i摘要 --- p.ivACKNOWLEDGEMENT --- p.viLIST OF PUBLICATIONS --- p.viiiABBREVIATIONS --- p.xiiiLIST OF FIGURES AND TABLES --- p.xviChapter 1 --- CHAPTER I: Introduction --- p.1Chapter 1.1 --- The ovary --- p.1Chapter 1.1.1 --- Structure and function of the ovary --- p.1Chapter 1.1.2 --- Follicle development --- p.5Chapter 1.1.3 --- Ovulation and luteinization --- p.7Chapter 1.1.4 --- Ovarian hormone biosynthesis --- p.10Chapter 1.2 --- Diseases with ovarian dysfunction --- p.14Chapter 1.2.1 --- Polycystic ovarian syndrome (PCOS) --- p.14Chapter 1.2.1.1 --- Introduction to PCOS --- p.14Chapter 1.2.1.2 --- Diagnostic criteria --- p.14Chapter 1.2.1.3 --- Abnormal hormone profile in PCOS --- p.16Chapter 1.2.1.4 --- Abnormal folliculogenesis in PCOS --- p.18Chapter 1.2.1.5 --- Etiology --- p.22Chapter 1.2.2 --- Cystic Fibrosis (CF) --- p.24Chapter 1.2.2.1 --- Introduction to CF --- p.24Chapter 1.2.2.2 --- Cause and pathogenesis of CF --- p.25Chapter 1.2.2.3 --- Ovarian disorder in CF --- p.27Chapter 1.3 --- CFTR in reproduction --- p.29Chapter 1.3.1 --- Introduction to CFTR --- p.29Chapter 1.3.2 --- Channel function --- p.30Chapter 1.3.3 --- Protein regulator function --- p.32Chapter 1.3.4 --- Regulation of CFTR expression --- p.34Chapter 1.3.5 --- Role of CFTR in reproduction --- p.35Chapter 1.3.6 --- CFTR in the ovary --- p.39Chapter 1.4 --- General hypothesis and aims --- p.39Chapter 1.4.1 --- General hypothesis --- p.39Chapter 1.4.2 --- Aims of the study --- p.40Chapter 2 --- CHAPTER II: General Methods --- p.42Chapter 2.1 --- Meterials --- p.42Chapter 2.1.1 --- Animals --- p.42Chapter 2.1.2 --- Chemicals and reagents --- p.42Chapter 2.1.3 --- Antibodies --- p.44Chapter 2.1.4 --- Primers --- p.45Chapter 2.2 --- Methods --- p.45Chapter 2.2.1 --- Determination of estrous cycle --- p.45Chapter 2.2.2 --- Granulosa cell culture --- p.46Chapter 2.2.3 --- PCGS rat model --- p.47Chapter 2.2.4 --- Collection of human granulosa cells --- p.47Chapter 2.2.5 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.48Chapter 2.2.6 --- Western blot --- p.50Chapter 2.2.7 --- Histological studies --- p.53Chapter 2.2.8 --- siRNA transfection --- p.55Chapter 2.2.9 --- Intracellular pH measurement --- p.56Chapter 2.2.10 --- Whole-cell patch clamp recording --- p.57Chapter 2.2.11 --- Statistics --- p.57Chapter 3 --- CHAPTER III: Result I - The Role of CFTR in FSH-stimulated Estrogen Production: Implication in Cystic Fibrosis and PCGS --- p.59Chapter 3.1 --- Summary --- p.59Chapter 3.2 --- Introduction --- p.60Chapter 3.3 --- Methods --- p.63Chapter 3.3.1 --- Intracellular cAMP assay --- p.63Chapter 3.3.2 --- Nuclei isolation and nuclear cAMP measurement --- p.63Chapter 3.3.3 --- CREB phosphorylation assay --- p.64Chapter 3.3.4 --- Estradiol enzyme immunoassay --- p.64Chapter 3.4 --- Results --- p.64Chapter 3.4.1 --- Functional expression of CFTR in granulosa cells --- p.64Chapter 3.4.2 --- Expression and localization of sAC in granulosa cells and its involvement in BC03f CFTR-dependent cAMP production --- p.66Chapter 3.4.3 --- Effect of CFTR and HC0₃⁻ on basal and FSB-stimulated CREB phosphorylation --- p.67Chapter 3.4.4 --- Effect of CFTR and HC0₃⁻ on basal and FSB-stimulated aromatase expression and estradiol production --- p.68Chapter 3.4.5 --- Impaired CREB phosphorylation aromatase expression and estradiol production by granulosa cells from CFTR-deficient mice --- p.70Chapter 3.4.6 --- Reduced CFTR and aromatase expression in human PCOS granulosa cells and rat PCOS ovaries --- p.71Chapter 3.5 --- Discussion --- p.87Chapter 4 --- CHAPTER IV: Result II - The Role of CFTR in Granulosa Cell Proliferation and survival in PCOS --- p.91Chapter 4.1 --- Summary --- p.91Chapter 4.2 --- Introduction --- p.92Chapter 4.3 --- Methods --- p.95Chapter 4.3.1 --- Cell viability assay (MTT and MTS assay) --- p.95Chapter 4.3.2 --- ERKI/2 phosphorylation assay --- p.95Chapter 4.4 --- Results --- p.96Chapter 4.4.1 --- Reduced CFTR expression in PCOS rat models --- p.96Chapter 4.4.2 --- Downregulation of genes related to proliferation and survival in PCOS --- p.96Chapter 4.4.3 --- CFTR affect viability of granulosa cells --- p.97Chapter 4.4.4 --- CFTR regulate cell cycle protein and promote proliferation via HC0₃⁻/sAC/PKA and ERK pathway --- p.98Chapter 4.4.5 --- CFTR regulates apoptosis-related protein expression --- p.100Chapter 4.5 --- Discussion --- p.114Chapter 5 --- CHAPTER V: General Discussion --- p.119Chapter 5.1 --- Role of CFTR in ovarian function --- p.119Chapter 5.2 --- Role of CFTR/HC0₃⁻/sAC in modulating FSH signaling in the ovary --- p.120Chapter 5.3 --- CFTR/HC0₃⁻/sAC as a general modulator in receptor-mediated signaling cascades --- p.122Chapter 5.4 --- Concluding remarks --- p.123REFERENCES --- p.124APPENDICES --- p.13