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LOCALIZATION and REGULATION of Mrna TRANSCRIPTS ENCODING ACTIVIN RECEPTORS in HUMAN PLACENTAL TROPHOBLAST CELLS

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LOCALIZATION and REGULATION of Mrna TRANSCRIPTS ENCODING ACTIVIN RECEPTORS in HUMAN PLACENTAL TROPHOBLAST CELLS LOCALIZATION AND REGULATION OF mRNA TRANSCRIPTS ENCODING ACTIVIN RECEPTORS IN HUMAN PLACENTAL TROPHOBLAST CELLS by Victor Chen B.M. Taipei Medical College, Taiwan, 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Obstetrics and Gynecology University of British Columbia We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA JUNE, 1997 ' ©Victor Chen, 1997 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of c9(rS^^Jy^c~>. frnd The University of British Columbia Vancouver, Canada Date DE-6 (2/88) ABSTRACT There is increasing evidence to suggest that activin can function as an autocrine/paracrine regulator in various tissues, including the reproductive system. At the cellular level, activin acts via a family of activin receptor (ActR) subtypes which includes two type I (ActRI and ActRLB) and two type II (II and ILB) receptors. The role of activin in the human placenta is not clearly understood. In this study, the detection of inlubin/activin subunit and ActRI mRNA were examined in first trimester cytotrophoblasts, term cytotrophoblasts, extravillous trophoblast (EVT) cells, immortalized extravillous trophoblast (LEVT) cells, JEG-3 cells, decidual tissue, and decidual cells. Regulation of ActRI mRNA levels was also studied in LEVT cells using competitive polymerase chain reaction (cPCR). With the exception of JEG-3 cells, ActRILB mRNA was detected in all cell populations, mRNA transcripts encoding inhibina, inhibinRA, inhibinpB, ActRI, ActRII and ActRILB were detected in the whole cell populations. However, ActRLB mRNA could not be detected in all cell populations. With Northern blot hybridization analysis, it was found that the ActRI mRNA level was greatest in first trimester EVT cells followed in decreasing order of first trimester cytotrophoblasts, JEG-3 cells and term cytotrophoblasts. An internal standard of ActRI cDNA for cPCR was constructed for the precise quantitation of the ActRI mRNA levels in LEVT cells. The ActRI mRNA accumulation was stimulated in a dose-dependent manner ii by activin-A with a maximal effect three times that of control culture at a dose of 10 ng/ml activin-A. The time course of the activin-A effects on ActRI mRNA levels showed an early response with the maximal increase at 6 hours. The effects of follistatin, an activin binding protein, with and without the concomitant presence of activin-A, were also examined. Follistatin had no effect on ActRI mRNA levels in the absence of activin- A. Co-treatment of activin-A and follistatin demonstrated the blocking effect of follistatin on activin-A with dose dependent manner. This study provides evidence for the first time that activin-A and follistatin modulate the ActRI mRNA levels in an autocrine/paracrine manner in human placental trophoblast cells. iii TABLE OF CONTENTS ABSTRACT ii LIST OF FIGURES vi LIST OF ABBREVIATION viii ACKNOWLEDGMENT x Chapter I: Introduction and Literature Review 1 A. Placenta 2 1. Introduction 2 2. Gross anatomy 4 3. Histology 5 4. Trophoblasts 8 a) differentiation 9 b) invasion 11 c) culture 13 5. Implantation 16 B. Growth factors 19 C. Activin and TGFp family 21 D. Follistatin 24 E. Activin receptors 25 E. Gene expression- 30 F. Competitive polymerase chain reaction 32 Chapter II. Objective 37 A. Background and rationale 37 B. Hypothesis and objective 39 Chapter III. Material and Methods 41 A. Material — 41 B. Methods 41 1. Cell preparation 41 2. Lnmunostaining 43 3. Cell culture 44 4. Hormonal treatment 45 5. RNA extraction 45 6. Northern blotting — 46 7. Reverse transcription 47 iv 8. Polymerase chain reaction and primers 47 9. Cloning 50 10. Mutagenesis 51 11. Validation 51 12. Competitive PCR- 53 13. Analysis 54 14. Statistics 54 Chapter IV. Results 55 A. Verification of the internal standard for optimizing cPCR. 55 B. Validation of cPCR. 57 C. The detection of activin subunit and ActRs mRNA in different cell populations using PCR. 57 D. Comparing ActRI mRNA levels in different cell populations using Northern blot analysis. 60 E. The effects of activin-A on the ActRI mRNA levels in cultured IEVT cells using cPCR. 60 F. The effects of follistatin on the ActRI mRNA levels in cultured IEVT cells using cPCR. 65 G. Interaction between follistatin and activin-A on the ActRI mRNA levels in cultured LEVT cells using cPCR. 65 H. The effects of inhibin, activin-A with inhibin, TGFpl, and GnRH on the ActRI mRNA levels in cultured LEVT cells using cPCR. 70 Chapter V. Discussion 72 Chapter VI. References 93 LIST OF FIGURES Figure 1. Common structural features of ActR Type I and II. 28 Figure 2. Schematic structure of the native and mutant ActRI cDNA used in PCR or cPCR. 52 Figure 3. Verification of the internal standard for optinrizing cPCR 56 Figure 4. Validation of cPCR (different concentration of native cDNA) 58 Figure 5. Validation of cPCR (different concentration of internal standard) 59 Figure 6. Comparing ActRI mRNA levels in different cell populations using Northern blot analysis 62 Figure 7. The effects of activin-A on ActRI mRNA levels in cultured LEVT cells using cPCR (Dose response). 63 Figure 8. The effects of activin-A on ActRI mRNA levels in cultured LEVT cells using cPCR (Time course). 64 Figure 9. The effects of follistatin on ActRI mRNA levels in cultured LEVT cells using cPCR (Dose response). 66 Figure 10. The effects of follistatin on the ActRI mRNA levels in cultured LEVT cells using cPCR. (Time course)-- 67 Figure 11. Interaction of follistatin and activin-A on ActRI mRNA levels in cultured LEVT cells using cPCR (Dose response). 68 Figure 12. Interaction of follistatin and activin-A on ActRI mRNA levels in cultured LEVT cells using cPCR vi (Time course). 69 Figure 13. The effects of GnRH, inhibin, inhibin plus activin-A, TGFpi on ActRI mRNA levels in cultured LEVT cells using cPCR. 71 vii LIST OF ABBREVIATION ActR activin receptor ActRI activin receptor type I ActRLB activin receptor type LB ActRII activin receptor type II ActRILB activin receptor type ILB BSA bovine serum albumin CAM cell adhesion molecule cAMP cyclic adenosine 5'-monophosphate Cdk2 cyclin-dependent protein kinase 2 cDNA complementary DNA cPCR competitive PCR CSF colony stimulating factor DEPC diethylene perchlorate DIG digoxigenin DNA deoxyribonucleic acid E2 estradiol ECM extracellular matrix EDTA emylenediaminetetraacetic acid EGF epidermal growth factor EVT extravillous trophoblast FBS fetal bovine serum FGF fibroblast growth factor FRP FSH releasing protein FS follistatin FSH follicle-stimulating hormone g gravitational constant GnRH gonadotropin releasing hormone GnRHR gonadotropin releasing hormone receptor hCG human chorionic gonadotropin LEVT immortalized extravillous trophoblast IGF insulin-like growth factor LL interleukin IS internal standard kDa kilodalton LH luteinizing hormone viii LIF leukemia irihibitory factor M Molar (mole/liter) MADR mothers against dpp-related proteins MMP metalloproteinase mRNA messenger RNA Q-PCR quantitative PCR progesterone P4 PAI plasminogen activator inhibitor PBS phosphate-buffered saline PCR polymerase chain reaction PDGF platelet-derived growth factor Q-PCR quantitative PCR Rb the product of the retinoblastoma gene RNA ribonucleic acid RT-PCR reverse transcription-PCR SDS sodium dodecyl sulfate SEM standard error of sample mean SSC sodium chloride and sodium citrate solution TBE Tris + Boric acid + EDTA TGFp transforming growth factor-beta TLMP tissue inhibitor of metalloproteinase TPR-I Receptors I of TGF-p TPR-n Receptors II of TGF-p VEGF vascular endothelial cell growth factor ix ACKNOWLEDGMENTS I would like to express my appreciation to my supervisor, Dr. Peter C.K. Leung, for his advice and support through my study. I would like to thank Dr. Chun Peng and Jessica Dy for assistance in the first part of this project. I would also like to thank Dr. Gregory Lee, Dr. Vugranam Venkatesh, and Dr. Colin MacCalman for serving on my supervisor committee and their helpful comments. I thank Dr. Lala for giving the immortalized extravillous trophoblast cells for research. I would also like to thank all members of Dr. Leung's laboratory for their support throughout my stay in the laboratory. Finally, I take this opportunity to express my deepest gratitude to my wife, Lilian Chen, for her love and continuing support throughout my academic endeavor. CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW Introduction Reproduction is controlled by the hypothalamus through a delicately designed network of gonadotropin-releasing hormone (GnRH) neurons that translate neural signals into endocrine messages to act on the anterior pituitary gland. The synthesis and secretion of GnRH is controlled by higher neural centers in the brain, allowing integration of a variety of influences. The pituitary hormones that regulate gonadal function are luteinizing hormone (LH) and follicle-stimulating hormone (FSH); these are synthesized in the gonadotropes in response to stimulation by GnRH. Gonadal hormones also directly regulate
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