by Wong, Cheuk-fai. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 95-99). / List of figures --- p.iv / List of tables --- p.v / Abbreviations --- p.vi / Abstract --- p.vii / Chapter I. --- INTRODUCTION --- p.1 / Chapter 1. --- Inhibin a brief review --- p.1 / Chapter 1.1 --- "Definition and nomenclature, including related substances" --- p.1 / Chapter 1.2 --- Structure --- p.2 / Chapter 1.3 --- Historical background of the inhibin concept --- p.4 / Chapter 1.4 --- Actions of inhibin --- p.9 / Chapter 1.5 --- Control of inhibin production --- p.10 / Chapter 1.6 --- Measurement --- p.11 / Chapter 1.6.1 --- Immunoassay --- p.11 / Chapter 1.6.2 --- Bioassay --- p.13 / Chapter 1.6.2.1 --- In vivo methods --- p.13 / Chapter 1.6.2.2 --- In vitro methods --- p.14 / Chapter 1.7 --- Inhibin in clinical studies --- p.15 / Chapter 2. --- Project design --- p.17 / Chapter 2.1 --- Background --- p.17 / Chapter 2.2 --- Objectives --- p.20 / Chapter II. --- MATERIALS AND METHODS --- p.21 / Chapter 1. --- Materials --- p.21 / Chapter 1.1 --- Tracer preparation and purification --- p.21 / Chapter 1.2 --- Inhibin RIA --- p.21 / Chapter 1.3 --- Other immunoassays --- p.22 / Chapter 2. --- In-house inhibin RIA development --- p.22 / Chapter 2.1 --- The tracer preparation --- p.22 / Chapter 2.2 --- The radioimmunoassay --- p.25 / Chapter 2.3 --- Optimization of assay parameters --- p.26 / Chapter 2.3.1 --- Optimization of serum content and second antibody titre --- p.26 / Chapter 2.3.2 --- Verification of second antibodies' precipitating activity --- p.27 / Chapter 2. --- INHIBIN-EASIA --- p.27 / Chapter 3. --- Progesterone --- p.28 / Chapter 4. --- "Oestradiol, LH, and FSH" --- p.28 / Chapter III. --- RESULTS --- p.30 / Chapter Part I. --- In-house inhibin assay development --- p.30 / Chapter 1. --- Iodination and purification --- p.30 / Chapter 1.1 --- Step 1:Iodination followed by Sephadex column purification --- p.30 / Chapter 1.2 --- Step 2: Red A gel column purification --- p.33 / Chapter 1.3 --- Step 3: Sephadex column purification --- p.33 / Chapter 2. --- Inhibin RIA: Binding and antibody dilution curve experiment --- p.36 / Chapter 3.1 --- Verification of binding activity of the second antibody --- p.38 / Chapter 3.2 --- Optimization of serum content/ second antibody titre --- p.39 / Chapter 4. --- Discussion and conclusion --- p.41 / Chapter Part II. --- Hormone results of women undergoing in vitro fertilization --- p.42 / Chapter 1. --- Presentation of analytical results --- p.42 / Chapter 2. --- Comparison of hormone profiles of patients in the two GnRH agonist regimes --- p.43 / Chapter 2.1 --- Gonadotropins --- p.43 / Chapter 2.2 --- E2 --- p.56 / Chapter 2.3 --- Progesterone --- p.62 / Chapter 2.4 --- Inhibin --- p.68 / Chapter 3. --- Relationship between hormone --- p.74 / Chapter 3.1 --- Relationship between hormone changes --- p.74 / Chapter 3.2 --- Regression analysis --- p.87 / Chapter IV. --- DISCUSSSION --- p.89 / Chapter 1. --- Hormone profiles --- p.89 / Chapter 2. --- Hormone correlation --- p.91 / Chapter V. --- CONCLUSION --- p.94 / Chapter VI. --- REFERENCES --- p.95 / Chapter VIII. --- Appendix 1 Protocol for study on IVF and inhibin --- p.100 / Chapter IX. --- Appendix 2 Protocol for the management of IVF cycles --- p.101 / Chapter X. --- Appendix 3 Patients' results (table) --- p.103 / Chapter XI. --- Appendix 4 Patients ' results (graph) --- p.110

In the present study, the effects of clomiphene citrate (CC) on ovulation, ovarian growth and ovarian steroidogenesis were examined. Ovulation in rats in response to PMSG was completely blocked by administration of three daily treatments of 1.0 mg CC/rat, but was restored by administration of hCG as a preovulatory LH surge substitute. When the number of treatment days was reduced to two days, 1.0 mg of CC enhanced ovulation in response to PMSG, whereas treatment for one day with the same dose of CC did not affect ovulation. The effects of CC on ovulation appear to be dose-dependent. The effects of CC on ovarian growth were similar to the effects of CC on ovulation. The ovarian growth induced by PMSG was inhibited by high doses of CC, while a lower dose had no effect. The inhibition of ovarian growth in terms of ovarian weight by a high dose of CC was restored by hCG given as a preovulatory LH surge. Treatment duration with CC appears to have an important influence on ovarian growth. Three daily treatments with high doses of CC significantly inhibited ovarian growth. However, when the number of treatment days was reduced from three to two, the opposite results were obtained in that CC significantly stimulated ovarian growth. The effects of CC on ovarian steroidogenesis in response to PMSG were dose-dependent. A higher dose of CC significantly stimulated estradiol-17β biosynthesis. Clomiphene citrate did not show any inhibitory effects on progesterone production. Progesterone production was stimulated by hCG in CC treated rats. Lower doses of CC stimulated progesterone and androgen production. Further studies on this are necessary. Histological examination of the ovary revealed that CC selectively inhibited the development of nondominant follicles. The dominant follicles were unaffected as for they were able to develop to the mature stage. These results suggest that the effects of CC on ovulation, ovarian growth and ovarian steroidogenesis are dose-dependent and affected by treatment duration. Clomiphene citrate is assumed to exert its action via a gonadotropic mechanism. / Medicine, Faculty of / Obstetrics and Gynaecology, Department of / Graduate

Tsang Lai-Ling Angel. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 136-145). / Abstracts in English and Chinese. / Abstract --- p.i / 論文撮要 --- p.iv / Acknowledgment --- p.vi / Table of Content --- p.vii / List of Publications --- p.xii / List of Figures --- p.xiv / List of Tables --- p.xvii / Abbreviations --- p.xviii / Chapter Chapter1 --- Introduction --- p.1 / Chapter 1.1 --- The Human Uterus Vs Rat Uterus --- p.1 / Chapter 1.1.1 --- Myometrium --- p.1 / Chapter 1.1.2 --- Endometrium --- p.1 / Chapter 1.2 --- The Human Endometrium Vs Rat Endometrium --- p.2 / Chapter 1.2.1 --- The structure of Human Endometrium --- p.2 / Chapter 1.2.2 --- Cyclic Changes in the Endometrium --- p.4 / Chapter 1.2.3 --- Physiological Roles of the Endometrium --- p.7 / Chapter 1.2.4 --- Uterine Fluid Volume and its Composition --- p.7 / Chapter 1.2.4.1 --- Regulation of Uterine Fluid Volume and Composition --- p.7 / Chapter 1.2.4.2 --- Role of Endometrial Epithelium in the Regulation of Uterine Fluid Volume --- p.9 / Chapter 1.3 --- Epithelial Ion Channels --- p.9 / Chapter 1.3.1 --- Epithelial CI- Channels in Secretory Epithelia --- p.11 / Chapter 1.3.1.1 --- Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) --- p.13 / Chapter 1.3.2 --- Epithelial Na+ Channel (ENaC) in Absorbing Epithelia --- p.18 / Chapter 1.3.3 --- ENaC and CFTR in Endometrial Epithelia --- p.26 / Chapter 1.4 --- Hormonal Regulation of the Endometrial Epithelium --- p.29 / Chapter 1.4.1 --- Estrogen and Progesterone --- p.29 / Chapter 1.4.2 --- Aldosterone --- p.32 / Chapter 1.5 --- Aim of Study --- p.35 / Chapter Chapter2 --- Materials and Methods --- p.38 / Chapter 2.1 --- Materials --- p.38 / Chapter 2.1.1 --- Culture Medium and Enzymes --- p.38 / Chapter 2.1.2 --- Drugs --- p.38 / Chapter 2.1.3 --- Molecular Biology --- p.39 / Chapter 2.1.4 --- Experimental Tissues and Animals --- p.39 / Chapter 2.2 --- Preparations --- p.39 / Chapter 2.2.1 --- Pervious Support for Cell Growth --- p.39 / Chapter 2.2.2 --- Growth Medium --- p.40 / Chapter 2.2.3 --- Culture of Mouse Endometrium Epithelial Cells --- p.43 / Chapter 2.2.4 --- Solutions for the Short-Circuit Current Measurement --- p.44 / Chapter 2.2.5 --- Electrodes for the Short-Circuit Current Measurement --- p.44 / Chapter 2.2.6 --- Solutions for Molecular Biology Experiment --- p.44 / Chapter 2.2.6.1 --- Diethyl Pyrocarbonate (DEPC)-treated Water --- p.44 / Chapter 2.2.6.2 --- lx TAE (DNA gel electrophoresis and its running buffer) --- p.45 / Chapter 2.2.6.3 --- 5x MOPS (RNA gel electrophoresis and its running buffer) --- p.45 / Chapter 2.2.6.4 --- Formaldehyde Gel-loading Buffer --- p.45 / Chapter 2.3 --- Protocols --- p.46 / Chapter 2.3.1 --- Effect of Ovarian Hormones and Aldosterone on CFTR and ENaC Expression --- p.45 / Chapter 2.3.2 --- Possible Interaction between CFTR and ENaC upon Hormones Stimulation --- p.47 / Chapter 2.4 --- Methods of Measurement --- p.48 / Chapter 2.4.1 --- The Short-Circuit Current Technique --- p.48 / Chapter 2.4.1.1 --- The Short-Circuit Current Setup --- p.48 / Chapter 2.4.1.2 --- Experimental Procedures --- p.52 / Chapter 2.4.1.3 --- Data Analysis --- p.55 / Chapter 2.4.2 --- Reverse Transcription - Polymerase Chain Reaction (RT-PCR) --- p.55 / Chapter 2.4.2.1 --- RNA Isolation --- p.55 / Chapter 2.4.2.2 --- RNA Gel Electrophoresis --- p.56 / Chapter 2.4.2.3 --- Reverse Transcription (RT) --- p.57 / Chapter 2.4.2.4 --- Primer used for the Polymerase Chain Reaction (PCR) --- p.58 / Chapter 2.4.2.5 --- General Procedure of PCR and Competitive RT-PCR --- p.59 / Chapter 2.4.2.6 --- DNA Gel Electrophoresis --- p.61 / Chapter 2.4.3 --- Capillary Electrophoresis - Laser Induced Fluorescence (CE-LIF) --- p.62 / Chapter 2.4.3.1 --- Capillary Tube --- p.54 / Chapter 2.4.3.2 --- Detection System --- p.65 / Chapter 2.4.3.3 --- Experimental Procedures --- p.65 / Chapter 2.4.3.4 --- Data Analysis --- p.66 / Chapter 2.4.4 --- Statistical Analysis / Chapter Chapter3 --- Results --- p.68 / Chapter 3.1 --- Influence of Ovarian Hormones on Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Epithelial Na+ Channel (ENaC) Expression in Mouse Endometrial Epithelium --- p.68 / Chapter 3.2 --- Culture Condition on Expression and Function of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Mouse Endometrial Epithelial Cells --- p.92 / Chapter 3.3 --- Expression Regulation of Endometrial Epithelial Na+ Channel (ENaC) Subunits and Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Na+ Diet During the Estrus Cycle in Mice --- p.98 / Chapter 3.4 --- Enhanced Epithelial Na+ Channel (ENaC) Activity in Mouse Endometrial Epithelium by Upregulation of γ-ENaC Subunit --- p.114 / Chapter Chapter4 --- General Discussion --- p.127 / Appendix --- p.132 / Chapter A. --- RNA Isolation --- p.132 / Chapter B. --- Reverse Transcription (RT) --- p.133 / Chapter C. --- Polymerase Chain Reaction (PCR) --- p.134 / Chapter D. --- Sequences and Conditions of All Primers --- p.135 / References --- p.136

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.iv / ACKNOWLEDGEMENT --- p.vi / LIST OF PUBLICATIONS --- p.viii / ABBREVIATIONS --- p.xiii / LIST OF FIGURES AND TABLES --- p.xvi / Chapter 1 --- CHAPTER I: Introduction --- p.1 / Chapter 1.1 --- The ovary --- p.1 / Chapter 1.1.1 --- Structure and function of the ovary --- p.1 / Chapter 1.1.2 --- Follicle development --- p.5 / Chapter 1.1.3 --- Ovulation and luteinization --- p.7 / Chapter 1.1.4 --- Ovarian hormone biosynthesis --- p.10 / Chapter 1.2 --- Diseases with ovarian dysfunction --- p.14 / Chapter 1.2.1 --- Polycystic ovarian syndrome (PCOS) --- p.14 / Chapter 1.2.1.1 --- Introduction to PCOS --- p.14 / Chapter 1.2.1.2 --- Diagnostic criteria --- p.14 / Chapter 1.2.1.3 --- Abnormal hormone profile in PCOS --- p.16 / Chapter 1.2.1.4 --- Abnormal folliculogenesis in PCOS --- p.18 / Chapter 1.2.1.5 --- Etiology --- p.22 / Chapter 1.2.2 --- Cystic Fibrosis (CF) --- p.24 / Chapter 1.2.2.1 --- Introduction to CF --- p.24 / Chapter 1.2.2.2 --- Cause and pathogenesis of CF --- p.25 / Chapter 1.2.2.3 --- Ovarian disorder in CF --- p.27 / Chapter 1.3 --- CFTR in reproduction --- p.29 / Chapter 1.3.1 --- Introduction to CFTR --- p.29 / Chapter 1.3.2 --- Channel function --- p.30 / Chapter 1.3.3 --- Protein regulator function --- p.32 / Chapter 1.3.4 --- Regulation of CFTR expression --- p.34 / Chapter 1.3.5 --- Role of CFTR in reproduction --- p.35 / Chapter 1.3.6 --- CFTR in the ovary --- p.39 / Chapter 1.4 --- General hypothesis and aims --- p.39 / Chapter 1.4.1 --- General hypothesis --- p.39 / Chapter 1.4.2 --- Aims of the study --- p.40 / Chapter 2 --- CHAPTER II: General Methods --- p.42 / Chapter 2.1 --- Meterials --- p.42 / Chapter 2.1.1 --- Animals --- p.42 / Chapter 2.1.2 --- Chemicals and reagents --- p.42 / Chapter 2.1.3 --- Antibodies --- p.44 / Chapter 2.1.4 --- Primers --- p.45 / Chapter 2.2 --- Methods --- p.45 / Chapter 2.2.1 --- Determination of estrous cycle --- p.45 / Chapter 2.2.2 --- Granulosa cell culture --- p.46 / Chapter 2.2.3 --- PCGS rat model --- p.47 / Chapter 2.2.4 --- Collection of human granulosa cells --- p.47 / Chapter 2.2.5 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.48 / Chapter 2.2.6 --- Western blot --- p.50 / Chapter 2.2.7 --- Histological studies --- p.53 / Chapter 2.2.8 --- siRNA transfection --- p.55 / Chapter 2.2.9 --- Intracellular pH measurement --- p.56 / Chapter 2.2.10 --- Whole-cell patch clamp recording --- p.57 / Chapter 2.2.11 --- Statistics --- p.57 / Chapter 3 --- CHAPTER III: Result I - The Role of CFTR in FSH-stimulated Estrogen Production: Implication in Cystic Fibrosis and PCGS --- p.59 / Chapter 3.1 --- Summary --- p.59 / Chapter 3.2 --- Introduction --- p.60 / Chapter 3.3 --- Methods --- p.63 / Chapter 3.3.1 --- Intracellular cAMP assay --- p.63 / Chapter 3.3.2 --- Nuclei isolation and nuclear cAMP measurement --- p.63 / Chapter 3.3.3 --- CREB phosphorylation assay --- p.64 / Chapter 3.3.4 --- Estradiol enzyme immunoassay --- p.64 / Chapter 3.4 --- Results --- p.64 / Chapter 3.4.1 --- Functional expression of CFTR in granulosa cells --- p.64 / Chapter 3.4.2 --- Expression and localization of sAC in granulosa cells and its involvement in BC03f CFTR-dependent cAMP production --- p.66 / Chapter 3.4.3 --- Effect of CFTR and HC0₃⁻ on basal and FSB-stimulated CREB phosphorylation --- p.67 / Chapter 3.4.4 --- Effect of CFTR and HC0₃⁻ on basal and FSB-stimulated aromatase expression and estradiol production --- p.68 / Chapter 3.4.5 --- Impaired CREB phosphorylation aromatase expression and estradiol production by granulosa cells from CFTR-deficient mice --- p.70 / Chapter 3.4.6 --- Reduced CFTR and aromatase expression in human PCOS granulosa cells and rat PCOS ovaries --- p.71 / Chapter 3.5 --- Discussion --- p.87 / Chapter 4 --- CHAPTER IV: Result II - The Role of CFTR in Granulosa Cell Proliferation and survival in PCOS --- p.91 / Chapter 4.1 --- Summary --- p.91 / Chapter 4.2 --- Introduction --- p.92 / Chapter 4.3 --- Methods --- p.95 / Chapter 4.3.1 --- Cell viability assay (MTT and MTS assay) --- p.95 / Chapter 4.3.2 --- ERKI/2 phosphorylation assay --- p.95 / Chapter 4.4 --- Results --- p.96 / Chapter 4.4.1 --- Reduced CFTR expression in PCOS rat models --- p.96 / Chapter 4.4.2 --- Downregulation of genes related to proliferation and survival in PCOS --- p.96 / Chapter 4.4.3 --- CFTR affect viability of granulosa cells --- p.97 / Chapter 4.4.4 --- CFTR regulate cell cycle protein and promote proliferation via HC0₃⁻/sAC/PKA and ERK pathway --- p.98 / Chapter 4.4.5 --- CFTR regulates apoptosis-related protein expression --- p.100 / Chapter 4.5 --- Discussion --- p.114 / Chapter 5 --- CHAPTER V: General Discussion --- p.119 / Chapter 5.1 --- Role of CFTR in ovarian function --- p.119 / Chapter 5.2 --- Role of CFTR/HC0₃⁻/sAC in modulating FSH signaling in the ovary --- p.120 / Chapter 5.3 --- CFTR/HC0₃⁻/sAC as a general modulator in receptor-mediated signaling cascades --- p.122 / Chapter 5.4 --- Concluding remarks --- p.123 / REFERENCES --- p.124 / APPENDICES --- p.138

INTRODUÇÃO: A estimulação ovariana, parte fundamental dos tratamentos de fertilização in vitro, baseia-se no conhecimento da fisiologia deste órgão. Recentemente um novo modelo de foliculogênese, a teoria das ondas foliculares, foi descrito em humanos. A sincronização do início da estimulação com o surgimento de uma onda folicular melhora o desempenho dos tratamentos em animais. Os protocolos de estimulação ovariana para fertilização in vitro humana não são sincronizados com o início de uma onda folicular. O presente estudo tem como objetivos avaliar duas estratégias de controle da emergência de uma onda folicular (aspiração do folículo dominante e indução da ovulação mediada pelo hCG) e descrever a estimulação ovariana sincronizada com o início de uma onda. MÉTODOS: Participaram deste estudo controlado pacientes com indicação de fertilização in vitro (fatores tubário e/ou masculino de infertilidade), randomizadas em três grupos: controle, \"hCG\" e \"aspiração\". No grupo controle (n=6), a aplicação do FSH recombinante (150UI/d) teve início no terceiro dia do ciclo menstrual, seguindo o protocolo flexível do antagonista do GnRH. Nos grupos experimentais foram realizadas ultrassonografias transvaginais seriadas até a identificação de um folículo dominante >= 15mm. Neste momento, no grupo \"hCG\" (n=5) foi aplicada dose de 250ug de hCG recombinante e, após dois dias , retomado o seguimento ultrassonográfico seriado. No grupo \"aspiração\" (n=5) o folículo dominante e os subordinados > 10mm foram aspirados e o seguimento ultrassonográfico seriado foi retomado após um dia depois. Quando foi detectada o aumento do número de folículos < 10mm, caracterizando a emergência de uma onda folicular, iniciou-se a estimulação ovariana nos grupos experimentais seguindo o mesmo protocolo do grupo controle. Os embriões produzidos foram criopreservados para transferência posterior devido a assincronia endometrial. RESULTADOS: As duas intervenções resultaram na emergência de ondas foliculares em todas as mulheres, um dia após a aspiração do folículo dominante e dois dias após a aplicação do hCG. A dose total de gonadotrofinas utilizada, o tempo de estimulação, a variação dos níveis séricos de estradiol, a variação do número de folículos pequenos, médios e grandes durante o tratamento, o número de oócitos obtidos, a taxa de fertilização e o número de embriões de morfologia adequada e inadequada foram semelhantes nos três grupos. A velocidade de crescimento do maior folículo foi menor nos grupos experimentais até o 5o dia do estímulo, aumentando a partir daí. Os níveis séricos de progesterona foram maiores nos grupos experimentais a partir do quinto dia do estímulo até o final do tratamento. Não houve influência mecânica da presença do corpo lúteo na dinâmica folicular e nos desempenho laboratorial dos oócitos ipsilaterais. Oito pacientes realizaram transferências embrionárias e três apresentaram resultado positivo do betahCG, todas dos grupos experimentais. CONCLUSÕES: As intervenções propostas são capazes de desencadear a emergência de uma onda folicular, permitindo a sincronização do estímulo ovariano com a emergência de uma onda. O estímulo ovariano sincronizado com a emergência de uma onda folicular resultou na produção de embriões viáveis / INTRODUCTION: Ovarian stimulation, an important step of in vitro fertilization treatments, relies on the understanding of ovarian physiology. Recently, a new model of human folliculogenesis has been suggested, based on waves of coordinated follicular development. In animal studies, it has been shown that ovarian stimulation synchronized with the emergence of a follicular wave results in better treatment outcomes. Ovarian stimulation protocols for in vitro fertilization in humans are not synchronized with wave emergence. Therefore, the objectives of this study were to investigate two strategies to control a follicular wave emergence (aspiration of the dominant follicle and hCG mediated ovulation induction) and to describe the effects of synchronizing the beginning of stimulation with the start of a follicular wave. METHODS: Women with indications of in vitro fertilization due to tubal and/or male fator infertility were invited to participate in this controlled trial. Participants were randomized to the following groups: control, \"hCG\" and \"aspiration\". Patients on the control group (n=6) were submitted to the flexible GnRH antagonist protocol starting recombinant FSH administration (150 IU/d) on the third day of the menstrual cycle. Women on the experimental groups underwent serial transvaginal sonography until a dominant follicle >= 15 mm was identified. In the \"hCG\" group (n=5) 250ug of recombinant hCG was administered; serial transvaginal sonography was resumed two days later. In the \"aspiration\" group (n=5) the dominant and subordinated follicles larger than 10mm were aspirated; serial transvaginal sonography was resumed one day later. When a follicular wave emergence was detected (increase in the number of follicles < 10mm), patients were submitted to an ovarian stimulation protocol similar to the control group. Embryos were cryopreserved for future transfer due to endometrial asynchrony. RESULTS: A follicular wave emerged one day after the dominant follicle aspiration or two days after the administration of recombinant hCG in all women. Total dose of gonadotropins administered, stimulation length, variation of serum estradiol during stimulation, variation in the number of small, medium and large follicles during stimulation, number of oocytes harvested, fertilization rates and the number of embryos with adequate and inadequate morphology were similar in the three groups. The largest follicle growth rate was inferior for women in both experimental groups until day 5 of stimulation, increasing thereafter. Serum progesterone levels were superior in both experimental groups between the 5th day and the end of stimulation. The presence of the corpus luteum did not influence mechanically the follicular dynamics nor the laboratory performance of the ipsilateral oocytes. Eight patients have already been submitted to embryo transfers; three had a positive betahCG test, all from the experimental groups. CONCLUSIONS: Both interventions are able to induce a follicular wave emergence allowing the synchronization of ovarian stimulation. The synchronized ovarian stimulation resulted in the production of viable embryos

INTRODUÇÃO: A estimulação ovariana, parte fundamental dos tratamentos de fertilização in vitro, baseia-se no conhecimento da fisiologia deste órgão. Recentemente um novo modelo de foliculogênese, a teoria das ondas foliculares, foi descrito em humanos. A sincronização do início da estimulação com o surgimento de uma onda folicular melhora o desempenho dos tratamentos em animais. Os protocolos de estimulação ovariana para fertilização in vitro humana não são sincronizados com o início de uma onda folicular. O presente estudo tem como objetivos avaliar duas estratégias de controle da emergência de uma onda folicular (aspiração do folículo dominante e indução da ovulação mediada pelo hCG) e descrever a estimulação ovariana sincronizada com o início de uma onda. MÉTODOS: Participaram deste estudo controlado pacientes com indicação de fertilização in vitro (fatores tubário e/ou masculino de infertilidade), randomizadas em três grupos: controle, \"hCG\" e \"aspiração\". No grupo controle (n=6), a aplicação do FSH recombinante (150UI/d) teve início no terceiro dia do ciclo menstrual, seguindo o protocolo flexível do antagonista do GnRH. Nos grupos experimentais foram realizadas ultrassonografias transvaginais seriadas até a identificação de um folículo dominante >= 15mm. Neste momento, no grupo \"hCG\" (n=5) foi aplicada dose de 250ug de hCG recombinante e, após dois dias , retomado o seguimento ultrassonográfico seriado. No grupo \"aspiração\" (n=5) o folículo dominante e os subordinados > 10mm foram aspirados e o seguimento ultrassonográfico seriado foi retomado após um dia depois. Quando foi detectada o aumento do número de folículos < 10mm, caracterizando a emergência de uma onda folicular, iniciou-se a estimulação ovariana nos grupos experimentais seguindo o mesmo protocolo do grupo controle. Os embriões produzidos foram criopreservados para transferência posterior devido a assincronia endometrial. RESULTADOS: As duas intervenções resultaram na emergência de ondas foliculares em todas as mulheres, um dia após a aspiração do folículo dominante e dois dias após a aplicação do hCG. A dose total de gonadotrofinas utilizada, o tempo de estimulação, a variação dos níveis séricos de estradiol, a variação do número de folículos pequenos, médios e grandes durante o tratamento, o número de oócitos obtidos, a taxa de fertilização e o número de embriões de morfologia adequada e inadequada foram semelhantes nos três grupos. A velocidade de crescimento do maior folículo foi menor nos grupos experimentais até o 5o dia do estímulo, aumentando a partir daí. Os níveis séricos de progesterona foram maiores nos grupos experimentais a partir do quinto dia do estímulo até o final do tratamento. Não houve influência mecânica da presença do corpo lúteo na dinâmica folicular e nos desempenho laboratorial dos oócitos ipsilaterais. Oito pacientes realizaram transferências embrionárias e três apresentaram resultado positivo do betahCG, todas dos grupos experimentais. CONCLUSÕES: As intervenções propostas são capazes de desencadear a emergência de uma onda folicular, permitindo a sincronização do estímulo ovariano com a emergência de uma onda. O estímulo ovariano sincronizado com a emergência de uma onda folicular resultou na produção de embriões viáveis / INTRODUCTION: Ovarian stimulation, an important step of in vitro fertilization treatments, relies on the understanding of ovarian physiology. Recently, a new model of human folliculogenesis has been suggested, based on waves of coordinated follicular development. In animal studies, it has been shown that ovarian stimulation synchronized with the emergence of a follicular wave results in better treatment outcomes. Ovarian stimulation protocols for in vitro fertilization in humans are not synchronized with wave emergence. Therefore, the objectives of this study were to investigate two strategies to control a follicular wave emergence (aspiration of the dominant follicle and hCG mediated ovulation induction) and to describe the effects of synchronizing the beginning of stimulation with the start of a follicular wave. METHODS: Women with indications of in vitro fertilization due to tubal and/or male fator infertility were invited to participate in this controlled trial. Participants were randomized to the following groups: control, \"hCG\" and \"aspiration\". Patients on the control group (n=6) were submitted to the flexible GnRH antagonist protocol starting recombinant FSH administration (150 IU/d) on the third day of the menstrual cycle. Women on the experimental groups underwent serial transvaginal sonography until a dominant follicle >= 15 mm was identified. In the \"hCG\" group (n=5) 250ug of recombinant hCG was administered; serial transvaginal sonography was resumed two days later. In the \"aspiration\" group (n=5) the dominant and subordinated follicles larger than 10mm were aspirated; serial transvaginal sonography was resumed one day later. When a follicular wave emergence was detected (increase in the number of follicles < 10mm), patients were submitted to an ovarian stimulation protocol similar to the control group. Embryos were cryopreserved for future transfer due to endometrial asynchrony. RESULTS: A follicular wave emerged one day after the dominant follicle aspiration or two days after the administration of recombinant hCG in all women. Total dose of gonadotropins administered, stimulation length, variation of serum estradiol during stimulation, variation in the number of small, medium and large follicles during stimulation, number of oocytes harvested, fertilization rates and the number of embryos with adequate and inadequate morphology were similar in the three groups. The largest follicle growth rate was inferior for women in both experimental groups until day 5 of stimulation, increasing thereafter. Serum progesterone levels were superior in both experimental groups between the 5th day and the end of stimulation. The presence of the corpus luteum did not influence mechanically the follicular dynamics nor the laboratory performance of the ipsilateral oocytes. Eight patients have already been submitted to embryo transfers; three had a positive betahCG test, all from the experimental groups. CONCLUSIONS: Both interventions are able to induce a follicular wave emergence allowing the synchronization of ovarian stimulation. The synchronized ovarian stimulation resulted in the production of viable embryos

A nutrição está intimamente relacionada com a reprodução nos animais e ainda há muito a se estudar sobre essa inter-relação, principalmente porque ainda há muita controvérsia na literatura sobre este tema. Por exemplo, alguns estudos sugerem ser benéfico o uso do \"flushing\" nutricional na produção de embriões em ruminantes. Entretanto, a maioria dos trabalhos mostra resultados negativos da alta ingestão de matéria seca (IMS). Dessa forma, existe a necessidade em se avançar nos estudos comparando a fisiologia reprodutiva e produção de embriões em fêmeas sob diferentes níveis nutricionais. Foram utilizadas 33 vacas Nelores não lactantes recebendo um dos quatro tratamentos a seguir: Os grupos Mantença (M), Restrição (0,7M) e Alta IMS (1,5M) receberam a mesma dieta padrão variando a quantidade oferecida. O grupo Alta ingestão de energia (E), recebeu uma dieta com alto teor de amido, com a mesma energia do grupo 1,5M, porém, com a mesma IMS do grupo M. Todas as vacas passaram por todos os tratamentos, em um modelo crossover. Estes animais tiveram o ciclo estral sincronizado e acompanhamento ovariano diário, por meio de ultrassonografia até confirmação da ovulação. Posteriormente, nos dias 7 e 10 do ciclo estral foi avaliado o volume do corpo lúteo (CL). Concentrações circulantes de hormônios esteróides (progesterona [P4] e estradiol [E2]) foram mensuradas em dias estratégicos. Os animais foram submetidos à aspiração intrafolicular (OPU) por volta do dia 12 do ciclo estral e dois dias após a OPU, os animais foram superestimulados e avaliou-se a resposta superovulatória e a qualidade embrionária entre os grupos, assim como a taxa de prenhez de 273 receptoras após transferência em tempo fixo dos embriões vitrificados. As análises foram realizadas de acordo com o Proc GLIMMIX do SAS, com nível de significância de P < 0,05. Os resultados estão apresentados na forma de média dos quadrados mínimos ± erro padrão. Este estudo englobou duas áreas principais: fisiologia e concentrações hormonais (Experimento 1) e produção in vivo de embriões (Experimento 2). Apesar de, no Experimento 1, terem sido avaliadas 33 vacas nas quatro repetições, totalizando 132 sincronizações, houve apenas 75 ovulações, as quais foram consideradas para as análises subsequentes. A taxa de ovulação não variou entre os tratamentos (~57%). O folículo ovulatório foi maior conforme a IMS aumentou para a dieta padrão nos grupos 1,5M, M, 0,7M (14,4 ± 0,3a vs 12,8 ± 0,3b vs 12,0 ± 0,3c nos grupos 1,5M, M e 0,7M, respectivamente). Apesar disso, o E2 circulante não diferiu entre os grupos. O volume do CL no Dia 7 do ciclo estral foi semelhante entre os grupos, todavia o grupo 1,5M apresentou menor concentração de P4 circulante (2,4 ± 0,2 ng/mL) em relação aos grupos M e 0,7M (2,9 ± 0,2 vs 3,0 ± 0,3 ng/mL, respectivamente). No Experimento 2, a concentração de insulina plasmática foi maior no grupo E (8,7 ± 0,86 ?UI/mL) comparado com os grupos 0,7M (4,6 ± 0,87) e M (5,3 ± 0,85), não diferindo do grupo 1,5M (6,6 ± 0,9). Houve maior resposta superovulatória (CL) nas doadoras que receberam a dieta padrão, 0,7M (13,0 ± 1,3a); M (14,2 ± 1,2ª) e 1,5M (13,9 ± 1,2ª) comparado às alimentadas com alta energia (9,7 ± 1,2b), devido à correlação negativa entre resposta superovulatória e insulina (r = -0,32). Apesar disso, não houve diferença entre os grupos para o número total de ovócitos/embriões colhidos (~6 estruturas), embriões viáveis (~3 embriões), ou congeláveis (~2,7 embriões). Independente de tratamento, a insulina circulante ao início da superovulação, apresentou correlação negativa com o número de embriões viáveis (r = -0,22). A prenhez aos 23 e 53 dias após a transferência dos embriões não diferiu entre os tratamentos, entretanto, a insulina circulante das doadoras teve correlação negativa com a prenhez das receptoras aos 60 dias de gestação (r = -0,16). Em conclusão, diferentes níveis de IMS alteraram o tamanho das estruturas ovarianas e/ou as concentrações circulantes de hormônios esteróides. Apesar disso, houve um efeito muito discreto de dieta na resposta superovulatória e qualidade embrionária das vacas. Independente de tratamento, alta insulina circulante das doadoras foi associada a menor resposta superovulatória, número de embriões viáveis e prenhez das receptoras aos 60 dias de gestação. / Nutrition is closely related to reproduction and there is still much to learn about this relationship, mainly because there is so much controversy in the literature on this topic. For example, some studies propose beneficial use of nutritional \"flushing\" on embryo production in ruminants. However, most studies show negative results of high dry matter intake (DMI). Thus, there is a need to advance in studies comparing reproductive physiology and embryo production in females under different nutritional levels. A total of 33 non-lactating Nelore cows received one of the following treatments: Groups maintenance (M), Restriction (0.7M) and High DMI (1.5M) received the same standard diet with variations in the amount offered. The group High intake of energy (E) received a diet with high starch content, but the same amount of energy as group 1.5M, however, with the same DMI as Group M. All cows underwent all treatments in a crossover model. These animals had their estrus synchronized and ovarian dynamics was monitored daily by ultrasound until confirmation of ovulation. Subsequently, on days 7 and 10 of the estrous cycle the volume of the corpus luteum (CL) was estimated. Circulating concentrations of steroid hormones (progesterone [P4] and estradiol [E2]) were measured in strategic days. The cows underwent intrafollicular aspiration (OPU) on day 12 of the estrous cycle, and two days after OPU, the cows ovaries were supererstimulated. The superovulatory response and embryo quality was compared among groups, as well as pregnancy rate of 273 recipients after receiving fixed-time transfer of vitrified embryos. Analyses were performed according to the Proc GLIMMIX of SAS, with a significance level of P < 0.05. Results are presented as least squares means ± standard error. This study included two main areas: physiology and hormone concentrations (Experiment 1) and in vivo embryo production and pregnancy rate (Experiment 2). Although, in Experiment 1, 33 cows were evaluated in four repetitions, totaling 132 synchronizations, there were only 75 ovulations, which were considered for subsequent analyzes. The ovulation rate did not vary between treatments (~57%). The size of the ovulatory follicle incresed as the DMI of the standard diet increased (14.4 ± 0.3a vs 12.8 ± 0.3b vs 12.0 ± 0.3c for groups 1.5 M, M and 0.7M, respectively). Nevertheless, the circulating E2 did not differ between groups. The volume of the CL on Day 7 of the estrous cycle increased as the DMI of the standard diet increased, but the 1.5M group had lower circulating concentrations of P4 (2.4 ± 0.2 ng/mL) than groups M and 0.7M (2.9 ± 0.2 and 3.0 ± 0.3 ng/mL, respectively). In Experiment 2, plasma insulin concentration was higher in the E group (8.7 ± 0.86 ?IU/mL) compared with groups 0.7 M (4.6 ± 0.87) and M (5.3 ± 0.85) not differing from group 1.5M (6.6 ± 0.9). There was a higher superovulatory response (CL) in donors receiving the standard diet [0.7M (13.0 ± 1.3), M (14.2 ± 1.2) and 1.5M (13.9 ± 1.2)] compared with those fed high energy (9.7 ± 1.2 CL) due to the negative correlation between superovulatory response and circulating insulin (r = - 0.32). Nevertheless, there was no difference between groups for the total number of oocytes/embryos collected (~6 structures), viable embryos (~3 embryos), or freezable (~2.7 embryos). Regardless of treatment, circulating insulin at the beginning of superovulation, was negatively correlated with the number of viable embryos (r = -0.22). Pregnancies at 23 and 53 days after embryo transfer did not differ between treatments, however, circulating insulin of the donor cows had a negative correlation with pregnancy of recipients at 60 days of gestation (r = -0.16). In conclusion, different levels of DMI changed the size of ovarian structures and/or the concentrations of circulating steroid hormones. Nevertheless, there was a very minor effect of diet on superovulatory response and embryo quality in cows. Regardless of treatment, high circulating insulin of the donors was associated with lower superovulatory response, lower number of viable embryos and less pregnancies of recipients at 60 days of gestation.

A nutrição está intimamente relacionada com a reprodução nos animais e ainda há muito a se estudar sobre essa inter-relação, principalmente porque ainda há muita controvérsia na literatura sobre este tema. Por exemplo, alguns estudos sugerem ser benéfico o uso do \"flushing\" nutricional na produção de embriões em ruminantes. Entretanto, a maioria dos trabalhos mostra resultados negativos da alta ingestão de matéria seca (IMS). Dessa forma, existe a necessidade em se avançar nos estudos comparando a fisiologia reprodutiva e produção de embriões em fêmeas sob diferentes níveis nutricionais. Foram utilizadas 33 vacas Nelores não lactantes recebendo um dos quatro tratamentos a seguir: Os grupos Mantença (M), Restrição (0,7M) e Alta IMS (1,5M) receberam a mesma dieta padrão variando a quantidade oferecida. O grupo Alta ingestão de energia (E), recebeu uma dieta com alto teor de amido, com a mesma energia do grupo 1,5M, porém, com a mesma IMS do grupo M. Todas as vacas passaram por todos os tratamentos, em um modelo crossover. Estes animais tiveram o ciclo estral sincronizado e acompanhamento ovariano diário, por meio de ultrassonografia até confirmação da ovulação. Posteriormente, nos dias 7 e 10 do ciclo estral foi avaliado o volume do corpo lúteo (CL). Concentrações circulantes de hormônios esteróides (progesterona [P4] e estradiol [E2]) foram mensuradas em dias estratégicos. Os animais foram submetidos à aspiração intrafolicular (OPU) por volta do dia 12 do ciclo estral e dois dias após a OPU, os animais foram superestimulados e avaliou-se a resposta superovulatória e a qualidade embrionária entre os grupos, assim como a taxa de prenhez de 273 receptoras após transferência em tempo fixo dos embriões vitrificados. As análises foram realizadas de acordo com o Proc GLIMMIX do SAS, com nível de significância de P < 0,05. Os resultados estão apresentados na forma de média dos quadrados mínimos ± erro padrão. Este estudo englobou duas áreas principais: fisiologia e concentrações hormonais (Experimento 1) e produção in vivo de embriões (Experimento 2). Apesar de, no Experimento 1, terem sido avaliadas 33 vacas nas quatro repetições, totalizando 132 sincronizações, houve apenas 75 ovulações, as quais foram consideradas para as análises subsequentes. A taxa de ovulação não variou entre os tratamentos (~57%). O folículo ovulatório foi maior conforme a IMS aumentou para a dieta padrão nos grupos 1,5M, M, 0,7M (14,4 ± 0,3a vs 12,8 ± 0,3b vs 12,0 ± 0,3c nos grupos 1,5M, M e 0,7M, respectivamente). Apesar disso, o E2 circulante não diferiu entre os grupos. O volume do CL no Dia 7 do ciclo estral foi semelhante entre os grupos, todavia o grupo 1,5M apresentou menor concentração de P4 circulante (2,4 ± 0,2 ng/mL) em relação aos grupos M e 0,7M (2,9 ± 0,2 vs 3,0 ± 0,3 ng/mL, respectivamente). No Experimento 2, a concentração de insulina plasmática foi maior no grupo E (8,7 ± 0,86 ?UI/mL) comparado com os grupos 0,7M (4,6 ± 0,87) e M (5,3 ± 0,85), não diferindo do grupo 1,5M (6,6 ± 0,9). Houve maior resposta superovulatória (CL) nas doadoras que receberam a dieta padrão, 0,7M (13,0 ± 1,3a); M (14,2 ± 1,2ª) e 1,5M (13,9 ± 1,2ª) comparado às alimentadas com alta energia (9,7 ± 1,2b), devido à correlação negativa entre resposta superovulatória e insulina (r = -0,32). Apesar disso, não houve diferença entre os grupos para o número total de ovócitos/embriões colhidos (~6 estruturas), embriões viáveis (~3 embriões), ou congeláveis (~2,7 embriões). Independente de tratamento, a insulina circulante ao início da superovulação, apresentou correlação negativa com o número de embriões viáveis (r = -0,22). A prenhez aos 23 e 53 dias após a transferência dos embriões não diferiu entre os tratamentos, entretanto, a insulina circulante das doadoras teve correlação negativa com a prenhez das receptoras aos 60 dias de gestação (r = -0,16). Em conclusão, diferentes níveis de IMS alteraram o tamanho das estruturas ovarianas e/ou as concentrações circulantes de hormônios esteróides. Apesar disso, houve um efeito muito discreto de dieta na resposta superovulatória e qualidade embrionária das vacas. Independente de tratamento, alta insulina circulante das doadoras foi associada a menor resposta superovulatória, número de embriões viáveis e prenhez das receptoras aos 60 dias de gestação. / Nutrition is closely related to reproduction and there is still much to learn about this relationship, mainly because there is so much controversy in the literature on this topic. For example, some studies propose beneficial use of nutritional \"flushing\" on embryo production in ruminants. However, most studies show negative results of high dry matter intake (DMI). Thus, there is a need to advance in studies comparing reproductive physiology and embryo production in females under different nutritional levels. A total of 33 non-lactating Nelore cows received one of the following treatments: Groups maintenance (M), Restriction (0.7M) and High DMI (1.5M) received the same standard diet with variations in the amount offered. The group High intake of energy (E) received a diet with high starch content, but the same amount of energy as group 1.5M, however, with the same DMI as Group M. All cows underwent all treatments in a crossover model. These animals had their estrus synchronized and ovarian dynamics was monitored daily by ultrasound until confirmation of ovulation. Subsequently, on days 7 and 10 of the estrous cycle the volume of the corpus luteum (CL) was estimated. Circulating concentrations of steroid hormones (progesterone [P4] and estradiol [E2]) were measured in strategic days. The cows underwent intrafollicular aspiration (OPU) on day 12 of the estrous cycle, and two days after OPU, the cows ovaries were supererstimulated. The superovulatory response and embryo quality was compared among groups, as well as pregnancy rate of 273 recipients after receiving fixed-time transfer of vitrified embryos. Analyses were performed according to the Proc GLIMMIX of SAS, with a significance level of P < 0.05. Results are presented as least squares means ± standard error. This study included two main areas: physiology and hormone concentrations (Experiment 1) and in vivo embryo production and pregnancy rate (Experiment 2). Although, in Experiment 1, 33 cows were evaluated in four repetitions, totaling 132 synchronizations, there were only 75 ovulations, which were considered for subsequent analyzes. The ovulation rate did not vary between treatments (~57%). The size of the ovulatory follicle incresed as the DMI of the standard diet increased (14.4 ± 0.3a vs 12.8 ± 0.3b vs 12.0 ± 0.3c for groups 1.5 M, M and 0.7M, respectively). Nevertheless, the circulating E2 did not differ between groups. The volume of the CL on Day 7 of the estrous cycle increased as the DMI of the standard diet increased, but the 1.5M group had lower circulating concentrations of P4 (2.4 ± 0.2 ng/mL) than groups M and 0.7M (2.9 ± 0.2 and 3.0 ± 0.3 ng/mL, respectively). In Experiment 2, plasma insulin concentration was higher in the E group (8.7 ± 0.86 ?IU/mL) compared with groups 0.7 M (4.6 ± 0.87) and M (5.3 ± 0.85) not differing from group 1.5M (6.6 ± 0.9). There was a higher superovulatory response (CL) in donors receiving the standard diet [0.7M (13.0 ± 1.3), M (14.2 ± 1.2) and 1.5M (13.9 ± 1.2)] compared with those fed high energy (9.7 ± 1.2 CL) due to the negative correlation between superovulatory response and circulating insulin (r = - 0.32). Nevertheless, there was no difference between groups for the total number of oocytes/embryos collected (~6 structures), viable embryos (~3 embryos), or freezable (~2.7 embryos). Regardless of treatment, circulating insulin at the beginning of superovulation, was negatively correlated with the number of viable embryos (r = -0.22). Pregnancies at 23 and 53 days after embryo transfer did not differ between treatments, however, circulating insulin of the donor cows had a negative correlation with pregnancy of recipients at 60 days of gestation (r = -0.16). In conclusion, different levels of DMI changed the size of ovarian structures and/or the concentrations of circulating steroid hormones. Nevertheless, there was a very minor effect of diet on superovulatory response and embryo quality in cows. Regardless of treatment, high circulating insulin of the donors was associated with lower superovulatory response, lower number of viable embryos and less pregnancies of recipients at 60 days of gestation.