Abstract 17β-Hydroxysteroid dehydrogenases (17HSDs) catalyze the reactions between 17-hydroxy and 17-keto steroids. In the present study, the enzyme activities and tissue distribution of 17HSD type 1, type 2 and type 4 were characterized. Furthermore, the role of 17HSD type 1 in estrogen-dependent growth was studied in MCF-7 breast cancer cells which were stably transfected with type 1 cDNA. Endogenous oxidative 17HSD activity found in COS-m6 monkey kidney cells was first compared with that of human placental 17HSD. Cultured COS-m6 cells exclusively possessed oxidative 17HSD activity, converting estradiol (E2) to less active estrone (E1). When placental 17HSD was transfected into these cells, highly reductive activity appeared. The 17HSD enzyme in COS-m6 cells also catalyzed the conversion of testosterone to androstenedione, whereas the placental enzyme was estrogen-specific. These results further proved the existence of different 17HSD isoenzymes. The enzymatic properties and cell- and tissue-specific expression of 17HSD type 1, type 2 and oxidative type 4 were further characterized. The data confirmed that in cultured cells the direction of 17HSD activity is determined by the expression of different isoenzymes and not by the intracellular environment. In addition, the 17HSD type 1 gene expresses two mRNA signals, 1.3 kb and 2.3 kb in size. The expression of 1.3 kb mRNA, but not 2.3 kb mRNA was related to enzyme concentration in all the cell types studied. The type 1 enzyme was expressed in the placenta, ovary and in some breast cancer specimens and in the cell lines originated from these tissues. 17HSD type 2 was more widely expressed in both steroidogenic and in target tissues of steroid action. 17HSD type 4 was expressed in almost all cell lines and in all tissues studied, but no correlation with 17HSD activity was detected. These results suggest that 17HSD type 1 is involved in E2 production in females and 17HSD type 2 is responsible for inactivation of sex steroids. However, the oxidation of 17β-hydroxysteroids seems not to be the primary activity of 17HSD type 4. The mRNAs for 17HSD type 1, type 2 and type 4 were found to be expressed in human mammary epithelial cells. In breast tissue samples both 17HSD type 1 and type 2 were detected by in situ hybridization. Despite the presence of 17HSD type 1 mRNA in human mammary epithelial cells, only oxidative 17HSD activity was detected. The reason for the lack of reductive activity is not yet known. Finally, MCF-7 breast cancer cells were stably transfected with 17HSD type 1 cDNA in order to study the effect of 17HSD type 1 on estrogen-dependent growth. In wild type MCF-7 cells, very low 17HSD activity was detected and E1 did not have any effect on cell growth. In the cells expressing 17HSD type 1, E1 was rapidly converted to E2. Hence in these cells E1 had a similar growth-promoting effect as E2 as a result of the action of 17HSD type 1. The presence of 17HSD type 1 in breast cancer cells may thus be an important factor regulating estrogen exposure and the estrogen-responsive growth of breast cancer tissue.

Diss. (sammanfattning) Stockholm : Karol. Inst., 2002. / Härtill 6 uppsatser.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 87-107).

Thesis (Ph. D.)--University of Missouri--Columbia, 2005. / "May 2005." Typescript. Vita. Includes bibliographical references (leaves 125-147). Also issued on the Internet.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2007. / Vita. Includes bibliographical references.

Ph.D.

Up to 80% of menopausal women suffer from hot flushes, consisting of a coordinated activation of heat loss mechanisms (sweating, cutaneous vasodilatation, etc.). Ovarian steroid withdrawal also leads to hypersecretion of gonadotropins (LH and FSH) and changes in body fat distribution. Because gonadotropin release, thermoregulation, and energy balance are hierarchically controlled by the hypothalamus, it is likely that changes in response to estrogen withdrawal are occurring at the level of the hypothalamus. The infundibular (arcuate) nucleus of the hypothalamus contains an estrogen-sensitive population of cells that co-express kisspeptin, neurokin B (NKB), and dynorphin ("KNDy neurons"). KNDy neurons have been proposed to be a site of estrogen negative feedback on gonadotropin release in multiple species because they are estrogen sensitive and respond to estrogen withdrawal with somatic hypertrophy and significant changes in gene expression. Because KNDy neurons project to known thermoregulatory centers in the hypothalamus (such as the median preoptic nucleus, MnPO), we also hypothesized that changes in thermoregulation were also a due to changes in KNDy neurons. Ovariectomized (OVX) rats also show disorders of thermoregulation, increased serum LH and FSH, and altered weight gain. Furthermore, OVX rats exhibit KNDy gene expression changes similar to changes seen in the human, making this model ideal to study the effects of estrogen withdrawal. We used a novel neurotoxin conjugate NK₃-SAP to ablate KNDy neurons in OVX female rats. We then observed core and tail skin temperatures, serum gonadotropin levels, and weight changes before and after replacement with 17β-estradiol. Next, we ablated NK3R-expressing neurons in the MnPO and monitored the thermoregulatory axis. Rats with KNDy-ablation did not exhibit the rise in LH and profound weight gain associated with ovariectomy. Furthermore, KNDy-ablated animals did not exhibit the chronic vasodilatation observed in OVX rats, providing the first evidence that KNDy neurons play a role in vasomotion. Rats with NK₃R cell-specific MnPO lesions also exhibited decreased activation of heat loss effectors. Together, these data demonstrate an important role for arcuate KNDy neurons in estrogen modulation of LH release and body weight, and demonstrate that NKB signaling is critical for activation of heat dissipation effectors.

Lee Mei Lan May. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 48-56). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 中文摘要 --- p.III / ACKNOWLEDGEMENTS --- p.IV / PUBLICATION --- p.V / LIST OF FIGURES --- p.VI / LIST OF TABLES --- p.VII / ABBREVIATION --- p.VIII / CONTENTS --- p.IX / Chapter CHAPTER ONE: --- INTRODUCTION AND LITERATURE / Chapter 1.1 --- THYROID CANCER AND ITS EPIDEMIOLOGY --- p.1 / Chapter 1.1.1 --- Histology --- p.1 / Chapter 1.1.2 --- Gender comparison --- p.3 / Chapter 1.1.3 --- Female hormone 一 risk factor --- p.6 / Chapter 1.2 --- BIOLOGICAL BACKGROUND OF HORMONE´-´ؤؤ --- p.7 / Chapter 1.2.1 --- Estrogen --- p.8 / Chapter 1.2.2 --- Estrogen antagonist --- p.9 / Chapter 1.2.3 --- Estrogen receptors --- p.10 / Chapter 1.2.4 --- Estrogen receptor and thyroid cancer --- p.12 / Chapter 1.3 --- THE ROLE OF APOPTOSIS --- p.13 / Chapter 1.3.1 --- Bcl-family protein --- p.13 / Chapter 1.3.2 --- Bcl-family protein and cancer --- p.14 / Chapter 1.3.3 --- Estrogen and Bcl-family protein --- p.15 / Chapter 1.4 --- Objectives --- p.16 / Chapter CHAPTER TWO: --- GENERAL MATERIALS AND METHODS / Chapter 2.1 --- MATERIALS --- p.17 / Chapter 2.1.1 --- Culture media and treatment reagents --- p.17 / Chapter 2.1.2 --- Reagents for Western blot assay --- p.18 / Chapter 2.1.3 --- Antibodies --- p.19 / Chapter 2.1.4 --- Materials for RT-PCR --- p.19 / Chapter 2.1.5 --- Kits --- p.20 / Chapter 2.1.6 --- Instrumentations --- p.20 / Chapter 2.2 --- Methods --- p.21 / Chapter 2.2.1 --- Cell culture and treatment --- p.21 / Chapter 2.2.2 --- MTT assay --- p.22 / Chapter 2.2.3 --- Western blot analysis --- p.23 / Chapter 2.2.3.1 --- Protein extraction --- p.23 / Chapter 2.2.3.2 --- SDS-PAGE and protein transfer --- p.23 / Chapter 2.2.3.3 --- Immunoblotting analysis --- p.24 / Chapter 2.2.4 --- RNA extraction and RT-PCR --- p.25 / Chapter 2.2.4.1 --- RNA extraction --- p.25 / Chapter 2.2.4.2 --- cDNA synthesis --- p.26 / Chapter 2.2.4.3 --- Polymerase Chain Reaction (PCR) --- p.27 / Chapter 2.2.5 --- Statistical analysis --- p.28 / Chapter CHAPTER THREE: --- RESULTS / Chapter 3.1 --- Effect of E2 and tamoxifen on proliferation --- p.29 / Chapter 3.2 --- Comparison of effects of E and testosterone on Proliferation --- p.31 / Chapter 3.3 --- Differential Bcl-xL expression in response to E2 and testosterone stimulation --- p.33 / Chapter 3.4 --- Expression of ERα and ERβ in response to E2 stimulation --- p.35 / Chapter 3.5 --- Bcl-xL and Bax protein expression in response to E2 stimulation --- p.36 / Chapter 3.6 --- Expression of Bcl-xL and Bax mRNA in response to E2 stimulation --- p.39 / Chapter CHAPTER FOUR: --- DISCUSSION --- p.41 / Chapter CHAPTER FIVE: --- CONCLUSION --- p.47 / REFERENCES --- p.48

Law, Ka Yan Jessica. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 194-203). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iv / Acknowledgements --- p.vi / Publications --- p.vii / Abbreviations --- p.viii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Mast Cell --- p.1 / Chapter 1.1.1 --- Origin of Mast Cell --- p.2 / Chapter 1.1.2 --- Heterogeneity of Mast Cell --- p.3 / Chapter 1.1.3 --- Activation of Mast Cells --- p.5 / Chapter 1.1.3.1 --- IgE-dependent Mast cell Activation --- p.5 / Chapter 1.1.3.2 --- IgE-independent Mast Cell Activation --- p.10 / Chapter 1.1.3.3 --- Importance of Calcium --- p.12 / Chapter 1.1.3.4 --- "Role of cyclic adenosine ´3ة,5'-monophosphate (cAMP)" --- p.15 / Chapter 1.1.4 --- Mast Cells Mediators --- p.16 / Chapter 1.1.5 --- Physiological and Pathological Roles of Mast Cells --- p.19 / Chapter 1.2 --- Oestrogens --- p.22 / Chapter 1.2.1 --- Oestrogens and Inflammation --- p.23 / Chapter 1.2.1.1 --- Pro-inflammatory Effects of Oestrogen --- p.23 / Chapter 1.2.1.2 --- Anti-inflammatory Effects of Oestrogen --- p.24 / Chapter 1.2.2 --- Oestrogen Signaling --- p.25 / Chapter 1.2.2.1 --- Classical Genomic Pathway --- p.26 / Chapter 1.2.2.2 --- Non-genomic Pathway --- p.30 / Chapter 1.3 --- Aims of Study --- p.34 / Chapter 2. --- Materials and Methods --- p.36 / Chapter 2.1 --- Materials --- p.36 / Chapter 2.1.1 --- Drugs --- p.36 / Chapter 2.1.1.1 --- Oestrogen Receptor Agonists and Antagonists --- p.36 / Chapter 2.1.1.2 --- DNA Transcription and Translation Blockers --- p.41 / Chapter 2.1.1.3 --- Mast Cell Secretagogues --- p.41 / Chapter 2.1.2 --- Materials for Histamine Assay Experiments --- p.41 / Chapter 2.1.2.1 --- Materials for Rat Sensitization --- p.41 / Chapter 2.1.2.2 --- Materials for Ovariectomy surgery --- p.42 / Chapter 2.1.2.3 --- Materials for Buffers --- p.42 / Chapter 2.1.2.4 --- Materials for Histamine Release Assay --- p.43 / Chapter 2.1.2.5 --- Miscellaneous --- p.43 / Chapter 2.1.3 --- Materials for Oestrogen Receptors Identification --- p.44 / Chapter 2.1.3.1 --- Oestrogen Receptors Expression --- p.44 / Chapter 2.1.3.1.1 --- Materials for RNA Extraction --- p.44 / Chapter 2.1.3.1.2 --- Materials for cDNA Construction by RT- PCR --- p.45 / Chapter 2.1.3.1.3 --- Materials for PCR --- p.45 / Chapter 2.1.3.1.4 --- Materials for Agarose Gel Electrophoresis --- p.46 / Chapter 2.1.3.1.5 --- Miscellaneous --- p.46 / Chapter 2.2 --- Histamine Assay Experiments --- p.47 / Chapter 2.2.1 --- Preparation of Buffers --- p.47 / Chapter 2.2.2 --- Preparation of Stock Solutions --- p.48 / Chapter 2.2.2.1 --- Preparation of Mast Cell Secretagogues Stock Solutions --- p.48 / Chapter 2.2.2.2 --- Preparation of Oestrogens Stock Solutions --- p.48 / Chapter 2.2.3 --- Animals and Rat Peritoneal Mast Cell Isolation --- p.49 / Chapter 2.2.3.1 --- Animals --- p.49 / Chapter 2.2.3.2 --- Rats Sensitization --- p.49 / Chapter 2.2.3.3 --- Ovariectomy Surgery --- p.49 / Chapter 2.2.3.4 --- Isolation of Rat Peritoneal Mast Cells --- p.50 / Chapter 2.2.3.5 --- Purification of Mast Cells --- p.51 / Chapter 2.2.3.6 --- Determination of Mast Cell Number and Viability --- p.51 / Chapter 2.2.4 --- Histamine Release Assay and Histamine Measurement --- p.52 / Chapter 2.2.4.1 --- Histamine Release Assay --- p.52 / Chapter 2.2.4.2 --- Fluorescence Assay of Histamine --- p.53 / Chapter 2.2.5 --- Calculation of Histamine Release Level and Inhibition --- p.54 / Chapter 2.2.6 --- Statistics Analysis --- p.55 / Chapter 2.3 --- Oestrogen Receptors Identification --- p.55 / Chapter 2.3.1 --- Oestrogen Receptors Expression --- p.55 / Chapter 2.3.1.1 --- Preparation of Buffers --- p.55 / Chapter 2.3.1.2 --- Isolation of Mast Cell mRNA --- p.56 / Chapter 2.3.1.3 --- Heparinase Treatment --- p.57 / Chapter 2.3.1.4 --- Construction of Mast Cell cDNA by RT-PCR --- p.57 / Chapter 2.3.1.5 --- Amplification of Oestrogen receptors cDNA by PCR method --- p.58 / Chapter 2.3.1.6 --- Agarose Gel Electrophoresis --- p.58 / Chapter 3. --- Effects of Oestrogens on Immunological Induced Histamine Release from Rat Peritoneal Mast Cells --- p.59 / Chapter 3.1 --- Introduction --- p.59 / Chapter 3.2 --- Materials and Methods --- p.62 / Chapter 3.3 --- Results --- p.63 / Chapter 3.3.1 --- Activation of RPMCs by Immunological Stimuli --- p.63 / Chapter 3.3.1.1 --- Effects of Oestrogen Receptor Agonists on Histamine Release from RPMCs --- p.63 / Chapter 3.3.1.2 --- Effects of Oestrogen on Anti-IgE mediated Histamine Release from RPMCs --- p.64 / Chapter 3.3.1.3 --- Effects of Selective Oestrogen Receptor Modulators on Anti-IgE Mediated Histamine Release from RPMCs --- p.65 / Chapter 3.3.1.4 --- Effects of Selective Oestrogen Agonists on Anti- IgE Mediated Histamine Release from RPMCs --- p.67 / Chapter 3.4 --- Discussion --- p.69 / Chapter 4. --- Elucidating the Mechanism Underlying the Effects of Oestrogens on Rat Peritoneal Mast Cells --- p.110 / Chapter 4.1 --- Introduction --- p.110 / Chapter 4.2 --- Materials and Methods --- p.115 / Chapter 4.3 --- Results --- p.116 / Chapter 4.3.1 --- The Involvement of Membrane Oestrogen Receptors --- p.117 / Chapter 4.3.1.1 --- Effects of Membrane Impermeable Oestrogen on Anti-IgE mediated Histamine Release from RPMCs --- p.117 / Chapter 4.3.2 --- Effects of Oestrogen Receptor Antagonists on the Modulating Actions of Oestrogen Receptor Agonists and SERMs on RPMC of Male Rats --- p.118 / Chapter 4.3.2.1 --- Effects on Oestrogen Receptor Agonists and SERMs Induced Low Level Histamine Release from RPMCs --- p.118 / Chapter 4.3.2.2 --- Effects on the Inhibitory Actions on Anti-IgE Mediated Histamine Release from RPMCs --- p.118 / Chapter 4.3.3 --- Effects of Transcription and Translation Blockers on the Inhibition Effects of Oestrogen Receptor Agonists and SERMs on RPMC of Male Rats --- p.122 / Chapter 4.3.4 --- Identification of Oestrogen Receptor Subtypes on RPMCs --- p.123 / Chapter 4.3.4.1 --- Molecular Expression of Oestrogen Receptor Subtypes on RPMCs --- p.123 / Chapter 4.3.5 --- Effects of Oestrogenic Agents on activation of RPMCs by Non- Immunological Stimuli --- p.123 / Chapter 4.3.5.1 --- Effects of Oestrogen on Compound48/80 Mediated Histamine Release from RPMCs --- p.123 / Chapter 4.3.5.2 --- Effects of Selective Oestrogen Receptor Modulators on Compound 48/80 Mediated Histamine Release from RPMCs --- p.124 / Chapter 4.3.5.3 --- Effects of Membrane Impermeable Oestrogen on Compound 48/80 Mediated Histamine Release from RPMCs --- p.125 / Chapter 4.3.5.4 --- Effects of Oestrogen on Substance P Mediated Histamine Release from RPMCs --- p.125 / Chapter 4.3.5.5 --- Effects of Selective Oestrogen Receptor Modulators on Substance P Mediated Histamine Release from RPMCs --- p.126 / Chapter 4.3.5.6 --- Effects of Membrane Impermeable Oestrogen on Substance P Mediated Histamine Release from RPMCs / Chapter 4.4 --- Discussion --- p.128 / References --- p.194

The present study was conducted to determine the effect of differential endogenous estrogen exposure in rats on stress-induced changes in spatial working memory. Subjects comprised male (n=8) and female (n=10) Sprague-Dawley rats, which were trained to complete a T maze, delayed alternation task. Performance was scored as a percentage of trials during which the correct maze arm was selected. Subjects scores were recorded after 1 and 2 hours of restraint stress, as well as after 1 hour of unimpeded movement in a cage placed in the testing room. Restraint stress was effected through physical confinement within plastic, cylindrical tubing. Female subjects underwent each of the testing conditions twice, during periods of high and low endogenous estrogen exposure, as ascertained by microscopic examination of vaginal epithelial cells for estrous cycle stage determination. Females in proestrus (elevated endogenous estrogen exposure) subjected to 1 hour of restraint performed significantly worse than their baseline scores (p=0.0017) or females in estrus (low endogenous estrogen exposure) after 1 hour of restraint (p=0.00014). After 1 hour of restraint, females in proestrus also committed an increased rate of perseverative errors compared to females in estrus, although this increase did not achieve statistical significance (p=0.06). No appreciable differences existed among subject groups in baseline performance or subsequent to 2 hours of restraint stress. Resultant data indicate impaired working memory among female rats under conditions of stress in the context of elevated endogenous estrogen exposure. This study, then, suggests a potential synergistic effect of stress and estrogen in compromising prefrontal cortex function and, therefore, may lend insight into the observed sex-related disparity in the incidence of major depressive disorder and other anxiety-related mood disorders.