REGULATED EXPRESSION OF CADHERIN-6 AND CADHERIN-11 IN HUMAN AND BABOON (PAPIO ANUBIS) ENDOMETRIUM by SPIRO GETSIOS B.Sc, McGill University, 1995 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Reproductive and Developmental Sciences Program Department of Obstetrics and Gynaecology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1997 © Spiro Getsios, 1997 Iii 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. 1 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 The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT: The human endometrium undergoes extensive proliferation and differentiation during the menstrual cycle. To date, the molecular mechanisms involved in the cyclic remodeling of the endometrium remain poorly characterised. The cadherins are a large family of integral membrane glycoproteins which mediate calcium-dependent cell adhesion and play a central role in the formation and organisation of tissues during development. We have recently determined that the two novel cadherin subtypes, cadherin-6 and cadherin-11, are present in the human endometrium. In view of these observations, we have examined the spatiotemporal expression of these two cadherin subtypes in this complex tissue. Cadherin-6 and cadherin-11 are expressed in the glandular epithelium during the proliferative phase. The expression of epithelial cadherin-6 declines as the cycle enters the secretory phase, whereas cadherin-11 levels in the glandular epithelium remain constant throughout the menstrual cycle. In contrast, these two cadherin subtypes are differentially expressed in the endometrial stroma. Cadherin-6 is only expressed in the proliferative endometrial stroma. The loss of cadherin-6 expression i n the stroma cells during the secretory phase is concomitant with an ii increase in the levels of cadherin-11. As the switch between cadherin- 6 and cadherin-11 in the endometrial stromal occurs when these cells are undergoing progesterone-mediated cellular differentiation, we examined the ability of this gonadal steroid to regulate these two endometrial cadherin subtypes in isolated endometrial stroma cells. Progesterone was capable of differentially regulating cadherin-6 and cadherin-11. In addition, we failed to detect cadherin-11 expression in endometrial biopsies obtained from women with habitual abortion associated with luteal phase deficiency, suggesting that cadherin-11 may play a central role in the functional maturation of the endometrium. Finally, we have localised these two cadherin subtypes in the baboon uterus in order to determine whether this non-human primate will serve as a suitable model in which to examine the role of cadherin-6 and cadherin-11 in implantation-related processes. The spatiotemporal expression of cadherin-6 and cadherin-11 in the human and baboon endometrium is similar. Collectively, these observations suggest that the two cadherin subtypes, cadherin-6 and cadherin-11, play a central role in the cyclic remodeling of the human endometrium in preparation for the implanting embryo. iii TABLE OF CONTENTS Abstract . ii Table of Contents iv List of Abbreviations vi List of Figures viii Acknowledgments ix 1: INTRODUCTION 1 1.1: The Human Endometrium , 2 1.1.1: Anatomy of the uterus 2 1.1.2: Cyclic remodeling of the endometrium during the menstrual cycle 3 1.1.3: Decidualisation 4 1.1.4: Human Implantation 6 1.1.5: Hormonal regulation of endometrial differentiation and receptivity 8 1.1.6: Growth factors in the human endometrium 12 1.1.7: Habitual abortion 14 1.2: Model Systems: 15 1.2.1: In vitro models of decidualisation 16 1.2.2: A model for human implantation: the baboon (Papio anubis) 17 1.3: Molecular Mechanisms Involved In Endometrial Remodeling And Human Implantation 18 1.3.1: The structure of the endometrial extracellular matrix 19 1.3.2: Matrix degradation 20 1.3.3: jntegrins 22 1.3.4: Cell-cell interactions.. 25 1.4: Cadherins 27 1.4.1: Classical cadherins 4 27 1.4.2: Structure of classical cadherins , 28 1.4.3: Cadherin-catenin interactions > 32 1.4.4: The cell biology of classical cadherins 35 1.4.5: The role of cadherins in the formation of intercellular junctional complexes and the establishment iv of epithelial cell polarity 37 1.4.6: The role of the cadherins in the maintenance of the differentiated non-invasive cell state 39 1.4.7: Regulation of classical cadherin expression 41 1.4.8: Type 2 (Atypical) cadherins 42 1.5: Cadherins and Human Implantation 44 1.5.1: Classical cadherins in the human endometrium and placenta 44 1.5.2: Type 2 cadherins in the human endometrium and placenta 45 1.6: Hypothesis and Rationale 47 2. MATERIALS AND METHODS: 50 2.1: Tissues 50 2.2: Cell Preparation and Culture 52 2.3: Immunohistochemistry , 53 2.4: Northern Blot Analysis 56 2.5: Western Blot Analysis 58 2.6: Statistical Analysis 59 3. RESULTS: 60 3.1: Localisation of cadherin-6 and cadherin-11 in the human endometrium during the menstrual cycle 60 3.2: Expression of the cadherin subtypes in the glandular epithelium and stroma of the human endometrium during the menstrual cycle 63 3.3: Differential regulation of cadherin-6 and cadherin-11 in cultured endometrial stroma cells by the gonadal steroid, progesterone 67 3.4: Cadherin-11 expression in the endometrium of women with habitual abortion associated with luteal phase deficiency 72 3.5: Localisation of cadherin-6 and cadherin-11 in the baboon (Papio anubis) uterus ...75 4. DISCUSSION: ...85 5. CONCLUSIONS: 103 References: 104 LIST OF ABBREVIATIONS ANOVA Analysis of variance APC Adenomatous polyposis coli BSA Bovine serum albumin °C degrees centigrade CaCI2 Calcium chloride cad cadherin CAM Cell adhesion molecule cAMP cyclic adenosine 3',5'-monophosphate CAR Cell adhesion recognition CAS Cadherin associated substrate cDNA complementary DNA CP cytoplasmic DMEM Dulbecco's Modified Eagle's medium DNA Deoxyribonucleic acid E2 17p-estradiol EC Extracellular ECL Enhanced chemiluminescence ECM Extracellular matrix EDTA Ethylenediamine tetra-acetate EGF Epidermal growth factor FCS Fetal calf serum FSH Follicle-stimulating hormone Fn Fibronectin g grams 9 gravity h hours hCG human Chorionic gonadotropin H202 Hydrogen peroxide IGF Insulin-like growth factor IGFBP-1 IGF binding protein-1 igG Immunoglobulin G kb kilobases kDa kiloDaltons Ln Laminin LiC03 Lithium carbonate MDCK Madine Darby canine kidney m i n minutes vi m I milliliter mM milli-Molar MMP Matrix metalloproteinase Mr Molecular weight mRNA messenger RNA MUC1 Episialin u,l microliter um micrometer u.M micro-Molar N-linked Asparagine-linked Na+,K+-ATPase Sodium, potassium-adenosine triphosphatase nM nano-Molar NaCI Sodium chloride p probability P4 Progesterone PA Plasminogen activator PAI-1 PA inhibitor-1 PBS Phosphate-buffered saline PGE2 Prostaglandin E2 PMSF Phenylmethyl sulfonyl flouride PRL Prolactin RNA Ribonucleic acid rRNA ribosomal RNA RT Room temperature RT-PCR Reverse transcriptase-polymerase chain reaction S Svedberg unit of flotation SDS Sodium dodecyl sulfate S E Standard error of the mean src Rouss sarcoma virus SSPE Standard saline phosphate-EDTA TGF-p Transforming growth factor-p TIMP Tissue inhibitor of MMP TM Transmembrane Tris-HCL Tris (hydroxymethyl)-aminomethane- Hydrochloric acid uPA Urokinase-type PA ZO Zonula occludens vii List of Figures Figure 1: Schematic representation of the structure of a cadherin molecule in the plasma membrane 29 Figure 2: Immunohistochemical localisation of cadherin-6 and cadherin-11 in the human endometrium at different stages of the menstrual cycle 61 Figure 3: Radioautogram of a Northern blot demonstrating the expression of E-cadherin, cadherin-6 and cadherin-11 in the glandular epithelium and stroma of the human endometrium at different stages of the menstrual cycle 65 Figure 4: Radioautogram of a Northern blot demonstrating the effects of progesterone on cadherin-6 and cadherin-11 mRNA levels in isolated human endometrial stroma cells 68 Figure 5: Western blot demonstrating the effects of progesterone on cadherin-6 and cadherin-11 expression in isolated human endometrial stroma cells 70 Figure 6: Immunohistochemical localisation of cadherin-11 in the secretory endometrium obtained from women with habitual abortion associated with luteal phase deficiency 73 Figure 7: Immunohistochemical localisation of cadherin-6 in the baboon endometrium at different stages of the menstrual cycle 77 Figure 8: Immunohistochemical localisation of cadherin-11 in the baboon endometrium at different stages of the menstrual cycle 79 Figure 9: Immunohistochemical localisation of cadherin-11 in the decidua of early pregnancy in the baboon 81 Figure 10: Immunohistochemical localisation of cadherin-6 and cadherin-11 in the baboon myometrium 83 Figure 11: Schematic representation of the correlation between the cyclic remodeling processes of the human endometrium and the expression of cadherin-6 and cadherin-11 86 VIII ACKNOWLEDGMENTS I would like to take this opportunity to express my indebtedness to Colin D. MacCalman for the immeasurable guidance and patience afforded me in these studies. In addition, I would like to extend my gratitude to Mary D. Stephenson for her enthusiastic support and providing endometrial biopsy specimens obtained from women with habitual abortion. I recognise the faculty members and students in the Dept. Ob/Gyn during the course of my studies and, in particular, the moral and intellectual motivation offered by George T.C. Chen. I would like to acknowledge A.T. Fazleabas for providing the baboon tissues. 1 would also like to thank Orest W.