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Regulated expression of cadherin-6 and cadherin-11 in human and baboon (Papio Anubis) endometrium Getsios, Spiro 1997

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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 cel ls 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 A b s t r a c t . ii Table of Contents iv List of Abb rev ia t i ons 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 A P C Adenomatous polyposis col i BSA Bovine serum albumin °C degrees centigrade CaCI 2 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 H 2 0 2 Hydrogen peroxide IGF Insulin-like growth factor IGFBP-1 IGF binding protein-1 igG Immunoglobulin G kb kilobases kDa kiloDaltons Ln Laminin L i C 0 3 Lithium carbonate MDCK Madine Darby canine kidney m i n minutes vi m I mi l l i l i te r mM mil l i -Molar MMP Matrix metalloproteinase Mr Molecular weight mRNA messenger RNA MUC1 Episialin u,l microli ter um micrometer u.M micro-Molar N- l i nked Asparagine-linked N a + , K + - A T P a s e 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 R T - P C R Reverse transcriptase-polymerase chain reaction S Svedberg unit of flotation SDS Sodium dodecyl sulfate S E Standard error of the mean s r c Rouss sarcoma virus SSPE Standard saline phosphate-EDTA TGF-p Transforming growth factor-p TIMP Tissue inhibitor of MMP TM Transmembrane T r i s - H C L 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. Blaschuk and ICOS Corp. for their kind gifts of reagents used in these studies. I am grateful for the financial support from the U.B.C. Graduate Fellowship for the duration of these studies. I would also like to extend a special thanks to D. Ashley Monks, George A. Hadjipavlou, Tanya Zenuk, and Claudia Giudici for providing an inspirational backdrop for these studies. Finally, I thank my parents, brother and sister for their endless support and encouragement. i x 1 INTRODUCTION: Infertility affects approximately 2.4 million couples in North America (1). An underlying cause of reproductive health problems such as recurrent pregnancy loss (2) and infertility (3) is believed to be implantation failure. Furthermore, despite increasing experience with assisted reproductive technologies, less than one in five women undergoing in vitro fertilization and embryo transfer establishes a viable pregnancy. A limiting factor in this setting may be the ability of the embryo to attach and subsequently invade into the endometrium. In order for successful implantation to occur, embryonic-uterine interactions must be initiated when the embryo and the endometrium have reached precise stages of development; the blastocyst must have acquired the ability to interact with the endometrium and the endometrium, in turn, must have undergone hormonally-dependent differentiation in preparation for the implanting embryo (4). To date, the molecular mechanisms which are involved in preparing the endometrium for the implanting embryo remain poorly characterised. I 1.1 The Human Endometr ium: 1.1.1 Anatomy of the uterus. The human uterus consists of three distinct tissue layers: the perimetrium, the myometrium and the endometrium (5). The perimetrium is a serosal layer which surrounds the uterus in the pelvic peritoneum. This continuous sheath is comprised of a single layer of mesothelial cells which are supported by loose connective tissue and contains the nerves which innervate the vascular and muscular components of the uterus. The muscular tissue layer of the uterus is termed the myometrium and consists of three poorly defined subdivisions of smooth muscle cells arranged in alternating orientations, each being interspersed with connective tissue and large blood and lymphatic vessels. The endometrium represents the mucosal lining of the uterus. Two cellular compartments comprise the endometrial tissue layer. The epithelial compartment consists of a simple columnar epithelium which can be further subdivided into a luminal and glandular epithelial subdomain. The endometrial stromal compartment is composed of multiple cell types, including fibroblastic stromal cells, bone marrow derived leukocytes and the smooth muscle and endothelial cel ls which constitute the spiral arteries found in this cellular component. The endometrium is often described as two functionally distinct tissue layers: the stratum functionalis and the stratum basalis. The stratum functionalis forms the major component of the endometrium and is actively shed and regenerated during the course of each menstrual cycle. The stratum basalis is a thin layer of progenitor cel ls which provides the materials and structural framework for the regeneration of the functionalis layer following menstruation. 1.1.2 Cyclic remodeling of the endometrium during the menstrual cyc le . The epithelial and stromal compartments of the human endometrium undergo cyclic proliferation, differentiation and shedding in response to the gonadal steroids, 17|3-estradiol (E2) and progesterone (P4) (6). After menstruation, the endometrium regenerates under the influence of E2 to produce a dense cellular stroma containing narrow tubular glands and small blood vessels. Following ovulation, in response to increasing levels of P4, glandular secretion and stromal 3 decidualisation become the predominant features of the endometrium. The P4-induced differentiation of the endometrial stroma during the secretory phase of the menstrual cycle results in a reduction in cellular density and the appearance of localised areas of edema. These changes become more pronounced as the secretory phase progresses. By the late secretory phase of the menstrual cycle, the spiral arteries of the endometrium become surrounded by cel ls resembling the terminally differentiated decidual cells of pregnancy. In the absence of pregnancy, P4 levels fall and the functionalis layer of the endometrium regresses and is shed during menstruation. However, if fertilisation occurs, P4 levels increase, causing extensive decidualisation of the endometrial stroma. To date, the molecular mechanisms which mediate the dynamic tissue remodeling processes that occur in the endometrium during each menstrual cycle remain poorly defined. 1.1.3 Dec idua l i sa t i on . In the human, decidualisation of the endometrial stroma occurs during the late secretory phase of the menstrual cycle even in the absence of pregnancy (6). The terminal differentiation of endometrial stroma cells into decidual cells is associated with both morphological and functional changes (7). Morphological decidualisation is expressed by a change to a polyhedral cell shape with an increase in cell size and by the extensive development of the organelles involved in protein synthesis (endoplasmic reticulum) and secretion (Golgi apparatus) (7,8). Other ultrastructural markers of decidualisation include the formation of desmosomes and gap junctions in the plasma membrane (9,10). Functionally, decidualisation is characterised by the onset of prolactin (PRL) (11) and insulin-like growth factor binding protein-1 (IGFBP-1) (12) secretion. Although the morphological and functional characteristics of decidual cells are well characterised, the sequence of molecular events involved in the differentiation of stromal fibroblasts into decidual cells remains poorly understood. The decidua plays an important role in the establishment and maintenance of pregnancy. Throughout pregnancy, the decidua cells are believed to fulfill paracrine, nutritional, immunoregulatory and embryoregulatory roles (13). In particular, the decidua cells are believed to regulate the invasive migration of embryonic trophoblast cells during human implantation (14,15). To date, the adhesive mechanisms involved in trophoblast-decidual cell interactions remain poorly characterised. 5 1.1.4 Human Implanta t ion . The first step in human implantation involves the apposition and attachment of the trophoectodermal layer of the blastocyst to the luminal epithelium of the endometrium (4,16). After this in i t ia l interaction, the embryonic trophoblast cells breach the epithelial barrier and invade into the endometrial stroma, to lie in intimate contact with the decidualised stroma cells. Subsequently, the trophoblast cells proliferate and differentiate into chorionic v i l l i which consist of two trophoblast cell layers: an inner layer of mitotically active mononucleate villous cytotrophoblast cells and an outer layer of the terminally differentiated syncytial trophoblast formed from the fusion of post-mitotic villous cytotrophoblasts. As pregnancy progresses, the cytotrophoblast cells at the tips of the v i l l i proliferate and breach the syncytial trophoblast layer to form extravillous cytotrophoblast columns. The extravillous cytotrophoblast columns are believed to anchor the placenta to the uterus (17). A subpopulation of extravillous cytotrophoblasts subsequently acquires a highly invasive phenotype which allows for these cells to dissociate from the column and invade deeply into the decidua, where they 6 penetrate the upper portion of the myometrium and the basal lamina of the uterine arterioles, thereby ensuring a continuous blood supply to the developing fetus (18). During human implantation, the extravillous cytotrophoblast cel ls must not only interact with one another but with the diverse cell types which constitute the human endometrium. Thus, trophoblast cel l invasion can be described as a precise series of membrane-mediated events (16). The adhesive mechanisms involved in the dynamic cellular interactions required for implantation in the human remain poorly understood. Trophoblast cell invasion mimics many of the events associated with tumour cell metastasis (19). However, unlike tumour cel l invasion, the migration of trophoblast cells in the endometrium is a highly regulated process. For example, the transplantation of murine blastocysts in extrauterine tissues such as the kidney (20), spleen (21) and test is (22), consistently increases the invasive capacity of trophoblast cells. These studies suggest that trophoblasts possess an intrinsic capacity for invasiveness and that the decidua is capable of limiting this invasion. In the human, errors in the depth of trophoblast invasion are associated with significant maternal and fetal morbidity 7 and mortality (23). For example, the absence of decidua allows for trophoblast cells to invade deep into the maternal tissues, as is the case in placenta accreta (24). In contrast, shallow invasion has been associated with preeclampsia, a disease which has extreme consequences on the health of both the mother and the fetus (25). The molecular mechanisms by which the decidua regulates trophoblast cel l invasion remain poorly characterised. 1.1.5 Hormonal regulation of endometrial di f ferent iat ion and recep t i v i t y . In the establishment of a successful pregnancy, the trophoblast cells of the blastocyst must interact with the endometrium during a defined period of the menstrual cycle, known as the window of implantation (4,26,27). Outside of this receptive period, the endometrium discourages interactions with the developing embryo. The existence of a window of implantation in the human was f i rs t suggested by the examination of secretory phase hysterectomy specimens for the presence of embryos (28). Embryos which had implanted in the endometrium were only observed after the twentieth day of a histologically defined 28 day menstrual cycle. Although 8 defining the precise time period of endometrial receptivity in the human has been a significantly difficult endeavor, embryo transfer data from women undergoing in vitro fertilisation indicate that the window of implantation in the human is approximately four days in duration, occurring between day ZO and 24 of the menstrual cycle (29). The role of gonadal steroids in regulating endometrial receptivity was first demonstrated in the rodent (26). In the E2-primed endometrium of ovariectomised rats treated with P4 for 4 days and then administered E2, the uterus became transiently receptive to the implanting blastocyst for up to 36 hours following the injection of E2 (26). Outside of this time period, the embryo would not implant. In addition, maintaining high levels of P4 prolonged this refractory period indefinitely. The effects of P4 on an E2-primed endometrium are considered to be paramount in preparing the endometrium for implantation. This is highlighted by recent findings demonstrating the ability of progesterone antagonists, such as RU-486 (also known as mifepristone) and ZK 98299 (also known as onapristone), to inhibit implantation in a l l species examined to date (30). The precise mechanism(s) by which RU-9 486 and other progesterone antagonists disrupt the cellular processes required for implantation remains an active area of investigation. The actions of E2 and P4 are mediated by their respective nuclear receptors (31). These receptors have been shown to be spatiotemporally expressed in the endometrium during the menstrual cycle (32-34). Under the influence of E2 in the proliferative phase, the number of E2 and P4 receptors increase in both the epithelial and stromal cells of the endometrium. In contrast, a reduction in the levels of these two steroid receptors is observed in the mid-secretory phase of the menstrual cycle, when P4 is the predominant steroid. The decrease in the levels of the P4 receptor appears to be more pronounced in the epithelial compartment, as the expression of this receptor in the stroma cells is maintained, albeit at lower levels when compared to the proliferative phase of the menstrual cycle (32). In agreement with these observations, E2 has been shown to up-regulate the number of E2 and P4 receptor whereas P4 has been shown to decrease E2 and P4 receptor levels in isolated human endometrial cel ls (35-39). In addition, failure to down-regulate P4 receptor levels in the endometrial epithelium has been correlated with primary infert i l i ty associated with luteal phase deficiency (40). 10 Although the role of the ovarian steroids, E2 and P4, in the differentiation of the endometrium during the menstrual cycle is wel l characterised, there are likely to be other factors which are important in mediating the decidualisation of the endometrial stroma. For example, androgens have been shown to be capable of maintaining, but not initiating, a decidual cell reaction in mice, even in the absence of P4 (41). Despite the presence of androgen receptors in human endometrial stromal and decidual cel ls (42), androgens are not capable of maintaining decidualisation in human endometrial stromal cel ls in vitro (43). Prostaglandins are also believed to be involved in the initiation and maintenance of decidualisation in the rodent and human (44,45). For example, human endometrial stroma cells treated with prostaglandin E2 (PGE2) and the ovarian steroids, E2 and P4, have been shown to have a synergistic effect on PRL secretion, a commonly used biochemical marker of decidualisation (45). In addition, the glandular epithelial cells of the human endometrium have been shown secrete high levels of PGE2, suggesting a paracrine relationship between the epithelium and stroma in mediating the process of decidualisation (46). Embryo-derived factors are also likely to mediate the early events associated with human implantation. For example, human u chorionic gonadotropin (hCG) receptors have been detected in human endometrial stroma cel ls (47), suggesting that hCG may play a direct role in the decidualisation of this cellular compartment. Furthermore, hCG has been shown to decidualise human endometrial stroma cells in vitro, even in the absence of gonadal steroids (48). However, PRL secretion was significantly increased when these primary cultures were treated with a combination of hCG and P4, suggesting that these two factors play a central role in decidualisation. Recent studies indicate that the effects of hCG and PGE2 on endometrial stroma cells are mediated by the intracellular secondary messenger, cycl ic adenosine 3',5'-monophosphate (cAMP) (49,50). 1.1.6 Growth factors in the human endometr ium. Growth factors have been shown to mediate, at least in part, the effects of gonadal steroids on the human endometrium (4,51). Several growth factors, including the insulin-like growth factors (IGF-I and -II) (52), epidermal growth factor (EGF) (53) and members of the transforming growth factor-p (TGFp) gene family (54), are spatiotemporally expressed in the endometrium. In the human endometrium, IGF-II mRNA levels are low in the proliferative phase of 12 the menstrual cycle but are readily detectable in late secretory endometrium and in the decidua of early pregnancy (52). Similarly, EGF immunostaining in the endometrial stroma is moderate in the proliferative phase of the menstrual cycle but is dramatically increased in the areas surrounding the spiral arteries (the areas of early decidualisation) in the late secretory phase of the menstrual cycle (53). Finally, TGFf5-l expression in the human endometrial stroma has been shown to increase in the secretory phase of the menstrual cycle (54). Taken together, these observations suggest that these three growth factors may play key roles in the differentiation of the endometrial stroma. Growth factors produced by the endometrium are also likely to play a central role in regulating the differentiation and invasive potential of trophoblast cells during human implantation (51). Since trophoblast cells lie at the maternal-fetal interface, they could be influenced by growth factors in either or both compartments. Several growth factor-receptor pairs have been described in human trophoblast cells. For example, these cells can both produce and respond to IGF-11 (53) and TGFp-l (55,56). IGF-II, which is produced by both the trophoblast and decidual cells, stimulates trophoblast cel l 13 invasion in primary cultures of first trimester extravillous cytotrophoblasts (55). In contrast, TGFf5-l has been shown to reduce the invasive capacity and can promote the differentiation of extravillous cytotrophoblasts in vitro (57). Furthermore, human cytotrophoblasts express receptors for EGF but are not able to produce the ligand which interacts with this receptor (58,59). EGF has been shown to induce syncytial trophoblast formation in cytotrophoblasts isolated from term placenta (58) and increase the invasive capacity of cytotrophoblasts isolated from the first trimester (59). It is therefore likely that TGFp-l and IGF-II, as well as maternally-derived EGF, play important roles in regulating trophoblast cell invasion, particularly in the early stages of pregnancy. However, the molecular mechanisms by which these growth factors regulate implantation-related processes remain poorly understood. 1.1.7 Habitual abor t ion : The maternal environment plays a critical role in both the establishment and maintenance of a pregnancy. Habitual abortion, defined as three or more consecutive spontaneous abortions, is believed to affect approximately 5% of couples trying to establish a family (60). 1 4 The causes of habitual abortion are multifactorial and include chromosomal, anatomical, endocrinological, and immunological factors (61). Luteal phase deficiency, defined as a maturational delay of at least three days in the endometrium, is believed to be an important cause of female infertility (3). Although recent studies indicate that the prevalence of luteal phase deficiency is substantially lower than was previously thought in infertile women (3,62), this diagnosis is still a major factor associated with habitual abortion (61). Recently, it has been postulated that an underlying deficiency in the endometrium may provide an environment that allows for the early stages of implantation to occur but is subsequently unable to support a viable fetal-placental unit (63). If such an abnormality were persistent, repeated pregnancy failure may occur. To date, a method for identifying an endometrium that is both receptive to the embryo and able to maintain a pregnancy, as opposed to exclusively a receptive endometrium remains to be identified. 1.2 Model Sys tems : 15 Progress in our understanding of human endometrial differentiation and embryo implantation has been slow. This is primarily due to the fact that in vivo human experimentation is not feasible and that these implantation-related processes are morphologically different in most experimental and domestic animals (64). Two model systems have recently emerged in which to examine the molecular and cellular processes of endometrial remodeling and implantation; in vitro culture systems established from human endometrial tissues and an in vivo model of human implantation using non-human primates. 1.2.1 In vitro models of dec idua l i sa t i on : Determining the factors which are involved in the regulation of endometrial cell differentiation in general, and in the decidualisation of the endometrial stroma in particular, has been hampered by the complexity of the hormonal and cellular interactions which occur in this tissue at the time of implantation. In order to address some of these issues, several in vitro models of decidualisation using isolated human endometrial stroma cells have been established (43,45,50,65). For example, endometrial stroma cells cultured in the presence of 16 physiological doses of E2 and P4 results in an induction in PRL and IGFBP-1 secretion, an increase in cell size and the formation of gap junctions (65,66). The extent to which the steroid-induced cellular differentiation observed in these primary cultures reflects the process of decidualisation in vivo has not been determined. 1.2.2 A model for human implantation: the baboon (Papio anubis). The use of the baboon as an experimental model for examining the cellular and molecular mechanisms associated with endometrial receptivity and embryo implantation has recently emerged as a potential alternative to primary cell cultures (67,68). The morphological and histological components of the non- human primate endometrium are analogous to the human and undergo simi lar remodeling processes during the reproductive cycle with one major exception (69); in the human endometrium, decidualisation occurs even in the absence of pregnancy (6). In contrast, the stromal compartment of the baboon endometrium initiates a predecidual change in the cel ls surrounding the spiral arteries during the secretory phase of the menstrual cycle but only undergoes extensive decidualisation in the 17 presence of an implanting embryo (69). For example, IGFBP-1, a frequently used marker for decidualisation in the human, is not detected in the baboon endometrial stroma in a non-conception cycle (70). However, IGFBP-1 is markedly up-regulated in this cellular compartment as it undergoes decidualisation during early pregnancy. Despite differences in the onset of decidualisation in the two species, a cr i t ica l role for the gonadal steroids, E2 and P4, has also been proposed in the regulation of endometrial remodeling in the baboon (67,71,72). As a result, the baboon may provide a useful model for studying implantation-related processes in vivo. 1.3 Molecular Mechanisms Involved In Endomet r ia l Remodeling And Human Implanta t ion: The extensive tissue remodeling processes which occur in the human endometrium in preparation for the implanting embryo are l ikely to involve complex changes in cell-cell and cell-matrix interactions. Cell-cell interactions not only play a role in the formation and organisation of the endometrium (73) and placenta (74,75), but may also mediate the diverse cellular interactions which occur between the trophoblast cells and the multiple cell types which constitute the 18 human endometrium (76). Cell-matrix interactions include the differentiation of the endometrial extracellular matrix (ECM) during the menstrual cycle (77,78), the remodeling of these matrices (production and breakdown) by the invading trophoblast cells during implantation (19) and the spatiotemporal expression of the molecules which mediate the interaction between cells of the placenta and the endometrial ECM (79,80). 1.3.1 The structure of the endometrial extracel lular ma t r i x . The ECM maintains tissue integrity and plays a key role in the regulation of cellular proliferation and differentiation (81). In the proliferative and early secretory phases of the menstrual cycle, the tightly associated stroma cells of the human endometrium are interspersed with a thin matrix that is primarily composed of fibronectin (Fn) and collagens type I, III, V and VI (77). At the onset of decidualisation, there is a marked reduction in collagen type VI subunit mRNA levels which coincides with the appearance of stromal edema in the endometrium (82). Concomitant with the decrease in collagen type VI transcripts is the de novo synthesis of laminins (Ln) type 2 and 4 (78). The decidual cells of early pregnancy produce a rich pericellular 19 matrix which includes both of these Ln subtypes and collagen type IV (83). Although the expression of Ln and collagen type IV is restricted to the stromal interstitium during the late secretory phase and in early pregnancy, the basement membrane of the endometrial epithelium and spiral arteries express these ECM components throughout the menstrual cycle (84). The biological significance of these changes in ECM production remain to be elucidated. The cytotrophoblast cells of the placenta also contribute to the endometrial ECM during implantation. For example, extravillous cytotrophoblasts have been shown to produce both Fn and Ln in vitro (85) . Both of these ECM components are found in close apposition with extravillous cytotrophoblast cells in vivo (85). In addition, an alternatively spliced variant of Fn, known as oncofetal Fn, is secreted by trophoblast cells and has been localised to the interface between the extravillous cytotrophoblast columns and the decidua in the human (86) . 1.3.2 Matrix degradat ion. The degradation of the ECM by proteases plays an important role in the remodeling of the endometrial stroma (43,54). 20 Two major classes of proteases, plasminogen activators (PA's) and matrix metalloproteinases (MMP's), have been detected in the human endometrium. The PA's are substrate-specific serine proteases involved in the cleavage of plasminogen to plasmin, but also exhibit a broad serine protease activity (87). Recent studies have demonstrated that cultured human endometrial stroma cells secrete urokinase-type PA (uPA) and that hormonally-induced decidualisation of these primary cultures significantly inhibits the secretion of this protease (88). The decrease in uPA secretion is concomitant with an up-regulation in the production of a uPA specific inhibitor, plasminogen activator inhibitor type 1 (PAI-1). The MMP's are a large family of zinc-dependent enzymes which are involved in a variety of tissue remodeling processes (89). Several MMP's have been shown to be spatiotemporally expressed in the human endometrium. For example, MMP-7, also known as matrilysin, is expressed in the glandular epithelium during the proliferative and late secretory phase, but is markedly down-regulated during the mid-secretory stage of the menstrual cycle (54). Similarly, the expression of the gelatinase, MMP-2, is dramatically increased in the stromal compartment of the late secretory phase and in decidualised stroma 21 cells cultured in the absence of P4 (43). Taken together, these observations suggest that proteases and their inhibitors play key roles in the differentiation and regression of the human endometrium. Proteinases have also been shown to play a central role in trophoblast cell invasion (19). For example, uPA activity has been detected in the mouse and human cytotrophoblasts (90,91). However, inhibiting uPA activity has no effect on trophoblast outgrowth or implantation in the mouse (92). In contrast, tissue inhibitors of MMP's (TIMP's) have been shown to inhibit human trophoblast cell invasion (91), whereas the gelatinase, MMP-9, appears to play a key role in potentiating the invasive capacity of these cells in vitro (93,94). Furthermore, the production of MMP-9 by trophoblast cells is down-regulated during the first trimester of pregnancy, paralleling the decline in trophoblast cell invasion observed with gestational age (23,95). 1.3.3 In tegr ins . Cell-ECM interactions are mediated by several classes of adhesion molecules, the best characterised of which are the integrins. The integrins are a large family of integral membrane glycoproteins 22 which are composed of heterodimeric a and p subunits and mediate both cell-ECM and cell-cell interaction (96). The ligand specificity of integrin heterodimers is determined by the particular combination of a and p subunits which are expressed on the cell surface. Integrin-mediated interactions with specific ECM ligands are thought to anchor the cell to the underlying substratum, provide the mechanical framework for cellular migration and activate several signal transduction pathways (96-98). The expression of several integrin subunits in the human endometrium are tightly regulated during the menstrual cycle. For example, the a1 , a4 and p3 integrin subunits are only expressed in the glandular epithelium during the secretory phase of the menstrual cycle, the timing of which correlates with the putative window of implantation (99,100). In contrast, the integrin subunits av and p i are expressed in the surface and glandular epithelium throughout the menstrual cycle. These observations have led to the proposal that the three integrin heterodimers, a l p l (collagen and Ln receptor), a4p l (Fn receptor) and avp3 (vitronectin receptor), can be used as molecular markers of endometrial receptivity (101). In the endometrial stroma, there is a marked increase in the expression of the p i integrin subunit 23 during decidualisation in vivo and in vitro, suggesting that this integrin subunit may also play a role in preparing the endometrium for the implanting embryo (102). Although the precise role(s) of these integrin subunits in human implantation have not been determined, a disruption in these expression patterns has been associated with primary infert i l i ty (103). The expression of integrin subunits is also highly regulated during trophoblast differentiation and invasion in the human (104,105). For example, the a6 and p4 integrin subunits have been localised to the villous cytotrophoblast cells of the human placenta (104). As these cells acquire an invasive phenotype, the expression of the a6p4 heterodimer (Ln receptor) is down-regulated and the cells begin to express the a5, a l , and p i subunits (105). Disruption of the a5p1 integrin subunit (Fn receptor) interactions, using function perturbing antibodies, has been shown to stimulate trophoblast cell invasion in vitro (105). In contrast, antibodies directed against the a l p l integrin subunits inhibits the invasive capacity of isolated trophoblast cells. In addition, interactions between integrin subunits and their ligands in vitro have been shown to stimulate tyrosine phosphorylation in human trophoblast cel ls (106). In view of these observations, i t has been 24 suggested that an elaborate equilibrium exists in trophoblast-ECM interactions which functions to both promote and restrain trophoblast invasion during implantation (105). These interactions may also provide the signaling environment necessary for regulating trophoblast cell differentiation. Alterations in the expression pattern of these integrin subunits have been observed in preeclampsia, a condition in which trophoblast cell invasion into the maternal tissues is compromised (107,108). In contrast, no differences in the expression of these integrin subunits were observed in the third trimester placenta of patients with severe preeclampsia, when the cl in ical features of this disease are clearly manifested (109). To date, i t remains unclear whether the aberrant expression of integrin subunits is the cause or merely a consequence of this disease of pregnancy. 1.3.4 Cel l -cel l i n t e rac t i ons . As the endometrial cells of the luminal epithelium are the f i rs t to interact with the trophectoderrh of the blastocyst, it is likely that specific cell adhesion molecules (CAMs) are expressed on the apical domain of these cells exclusively during the window of implantation. These molecules may mediate the initial attachment between the 25 trophoblast cells of the blastocyst and the endometrium. To date, the CAMs which are involved in the early events of human implantation have not been identified. , Recent studies have determined that several glycoproteins are spatiotemporally expressed in the endometrial epithelium during the menstrual cycle. For example, the expression of the cell surface glycoprotein, episialin (MUC1), is down-regulated during the receptive period in the mouse uterus (110). This mucin has been shown to inhibit integrin-mediated cell adhesion to the ECM (111), as well as ce l l -ce l l interactions (112). Taken together, these observations suggest that MUC1 may interfere with embryonic-uterine interactions and that the loss of this mucin from the cell surface may be involved in the acquisition of endometrial receptivity in the mouse. In contrast, MUC1 levels in the human endometrial epithelium are markedly increased during the putative window of implantation (113). Although these findings suggest that MUC1 may not play an anti-adhesive role during embryonic attachment in the human, aberrant expression of this mucin has been associated with habitual abortion (114). The biological significance of these observations remains to determined. To date, the adhesive mechanisms which mediate the cycl ic remodeling processes that occur in the endometrium in preparation for the implanting embryo remain poorly characterised. Furthermore, the CAMs involved in mediating the complex cellular interactions which occur between the trophoblasts and the multiple cell types which constitute the human endometrium have not been identified. 1.4 Cadher ins : The cadherins are a rapidly expanding gene superfamily of integral membrane glycoproteins which mediate calcium-dependent cell adhesion (115,116), Recent cloning studies suggest that this family of CAMs is comprised of two evolutionary distinct subfamilies: type 1 cadherins (also known as classical cadherins) and type 2 cadherins (also known as atypical cadherins) (117-119). 1.4.1 Classical cadher ins . The classical cadherins mediate cell adhesion in a homophilic manner (115,116). This subfamily includes the three originally identified cadherins: E-cadherin (E-cad), N-cadherin (N-cad) and P-cadherin (P-cad) (116,117). Consequently, these three classical 27 cadherin subtypes are the best characterised members of this large gene superfamily. 1.4.2 Structure of classical cadher ins . The classical cadherins consists of five extracellular subdomains, a single transmembrane domain and two cytoplasmic subdomains (Fig. 1) (120). Each of the first four extracellular subdomains contains the repeated amino acid motifs, DXNDN and DXD (D=aspart|c acid, N=aparagine and X=any amino acid), which are capable of binding calcium (121). These calcium binding sites are essential for cadherin mediated adhesion and are believed to stabi l ise the structural conformation of the cadherin extracellular subdomains (122). The first extracellular subdomain, which is the most distal from the plasma membrane, contains the classical cadherin cell adhesion recognition (CAR) amino acid sequence, HAV (H=histidine, A=alanine and V=valine) (123). Synthetic peptides which contain this conserved CAR sequence or antibodies generated against this amino acid sequence are capable of disrupting several cadherin dependent processes (124-127). 28 Figure 1: Schematic representation of the basic cadherin structure in the plasma membrane. The cadherins are comprised of f ive extracellular subdomains (EC1-EC5), a single transmembrane domain (TM) and two cytoplasmic domains (CP1 and CP2). The first four extracellular domains contain the acidic amino acid motifs, DXNDN and DXD (a and b, respectively), which have been shown to bind calcium. The CAR sequence, HAV, is present in the most distal extracellular domain (EC1) of classical cadherins. The cytoplasmic domains of the classical cadherins interact with a family of cytoskeletal-associated proteins, known as the catenins, which mediate the interaction between the cadherins and microfilaments of the cytoskeleton. The two cytoplasmic domains of the classical cadherins are the most highly conserved regions of these cell adhesion molecules. 29 I I | EC1 EC2 | EC3 EC4 | EC5 ™ CP11CP21 Ca2+ Ca** Ca*+ Ca*+ HAV ab ab ab ab EC1 EC2 EC3 EC4 ECS L TM catenins micro-filaments CP1 CP2 cell binding L j catenin binding - 1 N-cad E-cad P-cad TAPPY D TAPPY D TAPPY D SLLVFDYEGSGTAGSLSSLNSSSSGGEQDYDYLNDWGPR SUVFDYEGSGEMSLSSLNSSESDKDQOYOYLNEWGNR TLLVFOYEGSGSDAASLSSLTSSASOQDOOYDYLNEWGSR FKKLAD FKKLAD FKKLAD w o For example, peptides containing the HAV amino acid sequence inhibit neurite outgrowth, a process mediated by N-cad (125). In addition, antibodies generated against this CAR sequence are capable of disrupting Sertoli cell-germ cell interactions in vitro (127). The non-conserved amino acids which are immediately adjacent to the CAR site are thought to modulate the ability of the classical cadherins to interact with each other in a homophilic manner (128). The extracellular subdomain that is most proximal to the plasma membrane contains four cysteine residues which are thought to be involved in the formation of disulfide bridges and are conserved among the cadherins (115). Multiple N-linked glycosylatibn sites are also present in the extracellular domain of the classical cadherins (129). Recently, the three-dimensional structures of the binding regions of E-cad and N-cad, using two-dimensional nuclear magnetic resonance imaging (130) and X-ray crystallography (131), respectively, have been determined. The crystal structure for N-cad suggests that parallel molecules on the surface of the same cel l are able to dimerise prior to interacting with cadherins on an adjacent cel l membrane (130). This observation has led to the proposal that cadherin-mediated cel l adhesion is the result of a series of zipper-like interactions which 31 occur between identical cadherin subtypes on the surface of adjacent cell membranes. The transmembrane domain of the cadherins consists of a short sequence of hydrophobic amino acids arranged in an a -he l i ca l conformation (129). The two cytoplasmic domains are the most highly conserved regions among the classical cadherins (132). These domains interact with at least four intracellular proteins, known as a - , p - , ycatenin (also known as plakoglobin), and p120 C A S (Cadherin Associated Substrate) (132-134). The catenins mediate the interaction between the classical cadherins and the actin-based microfilaments of the cytoskeleton (135). 1.4.3 Cadherin-catenin i n t e rac t i ons . The classical cadherins cannot promote cell adhesion unless they are complexed with the catenins (135). The classical cadherins bind directly to either p - or y-catenin in a mutually exclusive manner (136). Recent studies indicate that the expression of these two catenins are differentially regulated during cellular differentiation and embryonic development (137,138). Cadherin-catenin complexes containing p -32 catenin are more abundant in relatively undifferentiated cells, whereas the number of complexes containing y-catenin increases with cellular differentiation (139). Furthermore, y-catenin cannot be substituted for p-catenin during the early embryonic development of p-catenin null mutant mice (140). Both of these cadherin-catenin complexes are believed to be involved in the formation of intercellular junctions. Recently, direct associations between the catenins and components of tight and desmosomal junctions have been described. For example, p-catenin binds directly to a tight junction associated protein, ZO-1 (141), whereas y-catenin has been shown to interact with desmoglein-1, a transmembrane component of desmosomes (142). Taken together, these studies indicate that p- and y-catenin mediate the formation of two structurally and functionally distinct cadherin-catenin complexes in the plasma membrane. a-catenin associates with both p- and y-catenin (143) and links both cadherin-catenin complexes to the cytoskeleton either directly (144) or indirectly via a-act in in (145). Recently, a novel member of the catenin gene family, known as pi 20 C A S , has been demonstrated to bind the cytoplasmic domain of classical cadherins independently of the other catenins (146). Although pi 20 C A S serves as a major substrate 33 for the src tyrosine kinase, the role of this novel catenin in the assembly of the cadherin-catenin complex remains to be determined. Phosphorylation of the catenins disrupts the stability of the cadherin-catenin complex, resulting in the disassembly of intercellular junctions (147). Activation of the oncoprotein p60 s r c (148) or ligand binding to the transmembrane tyrosine kinase receptor for EGF (149) results in the phosphorylation of p-catenin. In addition, p-catenin associates with a transmembrane protein tyrosine phosphatase which can dephosphorylate this cytoplasmic protein in vitro (150). These observations indicate that the catenins are downstream targets of intracellular signal transduction pathways. Furthermore, p-catenin mediates the interaction between the cadherin-catenin complex and the EGF receptor, suggesting that there is a direct link between cadherin function and receptor mediated signal transduction (151). The association of p120 C A S to the cadherin complex provides further evidence that the cadherins are involved in signal transduction cascades (134). Finally, p- and y-catenin have been shown to associate with the adenomatous polyposis coli gene product, APC, a product of a tumour suppressor gene which is frequently mutated in colorectal cancer (152). The organisation of these various catenin-containing 34 complexes are thought to regulate the pool of available catenins within the cytoplasm, thereby modulating cadherin function (153). 1.4.4 The cell biology of c lassical cadher ins . The classical cadherins are key morphoregulators (115,116,154). The spatiotemporal expression of these three cadherin subtypes is highly regulated during development: E-cad is first expressed in the mouse embryo at the one-cell stage (155), P-cad is first expressed in the mural trophectoderm of 4.5 day blastocysts (156) and N-cad is f i rs t expressed in the mesodermal cells of the gastrula (157). In general, the expression of the classical cadherins appears to be complementary during development. The homophilic specifications of cadherin-mediated interactions are thought to provide the molecular basis for the separation and segregation of embryonic cells during development, allowing for the formation and organisation of discrete tissues (116). For example, progenitor cells in the embryonic ectoderm which are destined to differentiate into neural tissues switch from expressing E-cad to N-cad (158,159). The switch in cadherin subtype expression allows for these N-cad expressing cells to separate from the ectodermal cel l layer and form the neural tube. Recent gene knock-out studies have highlighted the importance of the classical cadherins during development. A null mutation for the E-cad gene results in embryonic lethality in the mouse (160,161). E-cad-deficient embryos develop into abnormal blastocysts which cannot form trophectodermal epithelium. N-cad gene knock-out mouse embryos are surprisingly able to form neural tubes, but subsequently fail to form a yolk sac and exhibit both cardiac and neural abnormalities resulting in embryonic lethality (162). The presence of other unidentified cadherin subtypes may compensate for the loss of N-cad expression during the formation of the neural tube in the mouse. The expression of a particular cadherin subtype has been shown to govern the developmental fate of cells. For example, the transfection of either E-cad or N-cad cDNA into mutant murine embryonic stem cel ls lacking endogenous cadherin expression results in the formation of discrete tissue structures when injected into nude mice (163). E-cad promotes epithelial differentiation, whereas N-cad expression was consistent with the formation of neuroepithelial and cartilagenous structures. 36 The three classical cadherin subtypes have been localised to different tissues in the adult mouse (157). E-cad is expressed by epithelial, but not muscle or most neural cells. In contrast, N-cad has been localised primarily to muscle and neural cells. The only tissues in which murine P-cad is expressed in significant amounts are the placenta and decidua. This tissue distribution appears to be maintained in the human, with the exception of P-cad not being expressed in the human placenta and decidua (164,165). 1.4.5 The role of cadherins in the formation of i n t e r c e l l u l a r junctional complexes and the establishment of epithelial c e l l po la r i t y . The classical cadherins mediate the formation of the intercellular junctional complex in epithelial cel ls (including tight junctions, adherens junctions, desmosomal junctions, and gap junctions), before themselves being localised to the membrane domain of adherens junctions (166,167). For example, antibodies generated against E-cad have been shown to disrupt the formation of tight junctions and desmosomes in cultured Madine-Darby canine kidney (MDCK) cel ls (166). Similarly, antibodies generated against E-cad and 37 N-cad disrupt gap junction formation in keratinocytes (168) and cardiomyocytes (169), respectively. Finally, the reorganisation of gap junctions in hepatocytes following partial hepatectomy in mice are colocalised to foci of preformed cell-cell contacts containing E-cad-catenin complexes (170). Collectively, these observations suggest that the cadherins mediate an early adhesive event which is a prerequisite for the recruitment of the components of these intercellular junctions. Classical cadherins play a central role in the establishment of epithelial cell polarity (171,172). In particular, E-cad has been shown to be a key regulator of membrane-cytoskeletal interactions and protein distribution within the plane of the plasma membrane. E-cad restricts the distribution of the sodium, potassium-adenosine triphosphatase (Na+,K+-ATPase) to the basolateral membrane domain of polarised epithelial cells, including the cells of the mural trophectoderm of the mammalian blastocyst (173). The interaction between E-cadherin and the Na+,K+-ATPase may explain, at least in part, why E-cad null mutant mice fail to form a normal blastocoel in utero (160). In addition, transfection of fibroblast cells with the fu l l length E-cad cDNA causes the redistribution of the Na+,K+-ATPase to sites of E-cad mediated contact, whereas the transfection of E-cad 38 cDNA constructs lacking the catenin-binding domain does not restr ict the distribution of this enzyme (171). Collectively, these observations demonstrate that E-cad mediated cellular interactions initiate a cascade of molecular events that results in the assembly of a membrane complex composed of E-cad and the Na+,K+-ATPase. This complex is believed to be linked to the cytoskeleton by the act in-associated protein, known as fodrin (171). 1.4.6 The role of cadherins in the maintenance of the dif ferent iated non-invasive cell s t a te . E-cad is a key regulator of the differentiated non-invasive cel l state (174-176). For example, MDCK cells form tight cell monolayers which display many of the characteristics of polarised epithelia in vivo (177). In the presence of antibodies directed against E-cad, the intercellular junctional complexes disassemble and the MDCK cel ls acquire a fibroblastic phenotype (174). These fibroblast-like cel ls become motile and invade into collagen gels and embryonic chick heart tissue (174). Furthermore, the transfection of full-length E-cad cDNA into invasive human carcinoma cells not only decreases the invasive 39 potential of these cancer cells but also induces a reversion to the polarised epithelial cell state (178). The anti-invasive role of E-cad is not restricted to cells in culture. An inverse correlation between the levels of E-cad expression and the invasiveness of carcinoma cells has been described in numerous human cancers (179). In general, poorly differentiated, highly metastatic carcinoma cells express low or undetectable amounts of E-cad, whereas well differentiated* poorly metastatic carcinomas frequently express this CAM. These observations have led to the hypothesis that E-cad is the product of a tumour suppressor gene (179). The aberrant expression of other cadherin subtypes may also play a role in the neoplastic transformation of epithelial cel ls (180). For example, high levels of N-cad are present in invasive squamous carcinomas (180) and metastatic breast cancer cel ls (181). In addition, the transfection of full length N-cad cDNA into differentiated squamous epithelial cells disrupts cellular interactions and promotes a scattered fibroblastic phenotype in these cel ls (180). Finally, the inhibition of N-cad expression in poorly differentiated squamous carcinoma cel ls, using antisense oligonucleotides, causes these cel ls to revert to an epithelial phenotype. Collectively, these observations 40 suggest that the loss of E-cad and the aberrant expression of N-cad may promote an invasive phenotype in carcinoma cells. 1.4.7 Regulation of c lassical cadherin express ion . Gonadal steroids are key regulators of classical cadherin expression in mammalian tissues. For example, P4 and E2 (but not testosterone or dihydrotestosterone) are capable of increasing E-cad mRNA levels in the immature mouse uterus in vivo (182). In addition, E2 was found to be capable of increasing E-cad mRNA levels in the surface epithelium of the mouse ovary in vivo (183) and in human breast carcinoma cell lines in vitro (184). Gonadal steroids are also capable of regulating N-cad expression (185-187). For example, E2 is capable of regulating N-cad expression in rat granulosa cel ls in vitro (185) and N-cad mRNA levels in the immature mouse ovary and testis in vivo (186,187). The intracellular signaling molecule, cAMP, has also been shown to increase E-cad expression in trophoblasts isolated from human term placenta (188) and in dog and human thyrocytes in vitro (189). Similarly, Follicle-Stimulating Hormone (FSH) and its secondary messenger, cAMP, stimulate N-cad mRNA levels in isolated mouse 41 Sertoli cel ls (190). However, E2 and cAMP in combination, are required for maximal N-cad mRNA levels in the mouse Sertoli cell cultures. I. 4.8 Type 2 (Atypical) cadher ins . The type 2 cadherin subfamily, also known as the atypical cadherins, includes the human cadherin subtypes, cadherin-5, -6, -8, -I I , -12, -13, and -14 (117,118,191), as well as other cadherin subtypes identified in the rodent (192), chicken (193), and Xenopus (194). The type 2 cadherins, like the classical cadherins, are composed of five extracellular subdomains, a transmembrane domain, and two cytoplasmic subdomains (116-118). The calcium binding motifs found in the extracellular domain of the classical cadherins are also conserved among the type 2 cadherins. In addition, the type 2 cadherin subtypes share the well conserved catenin-binding cytoplasmic domain. Despite these similarities, the over-all amino acid sequence homology between these two subfamilies is low (<35%) (119). In addition, several characteristic amino acid deletions and substitutions are observed among the type 2 cadherin subtypes. In particular, the type 2 cadherins do not contain the classical cadherin CAR sequence, HAV. Although the CAR sequence for the type 2 cadherins has not been 42 identified, these cadherin subtypes have been shown to mediate calcium-dependent cell adhesion in a homophilic manner (193,195). For example, the transfection of L cells (a fibroblasti c cell line which does not express endogenous cadherins) with the full length cDNAs of different type 2 cadherins has been shown to induce cadherin subtype-specific cellular aggregation (193,195). The cell biology of the type 2 cadherins remains poorly characterised. However, there is increasing evidence that these CAMs play a central role in morphogenesis and tumourigenesis. For example, the spatiotemporal expression of cadherin-11 (cad-11 or OB-cad) is associated with bone formation (196) and the development of the central nervous system in the rodent embryo (195,197). In addition, cadherin-6 (cad-6 or K-cad) is believed to play a central role in the formation of the human (198,199) and rodent (200) kidney. The expression of this type 2 cadherin subtype has been detected in kidney and prostate carcinomas, suggesting that cad-6 may be involved in the neoplastic transformation of these cel ls (199,201,202). Finally, cad-6 has been localised to specific regions of the embryonic mouse brain (203). The spatiotemporal expression pattern of cad-6, cad-11, as wel l as another type 2 cadherin subtype, known as cadherin-8 (cad-8), in the 43 mouse brain suggests that the differential expression of type 2 cadherin subtypes plays a central role in organising neural structures during the development of the rodent central nervous system (204,205). 1.5 Cadherins and Human imp lan ta t i on : In view of the central role that the cadherins play in the formation and organisation of tissues during development as well as in maintaining tissue integrity in the adult, it would seem likely that members of this gene superfamily are involved in human implantation. 1.5.1 Classical cadherins in the human endometrium and p lacenta . The expression of the classical cadherins in the human endometrium and placenta have been previously described. E-cad and P -cad are expressed in the surface and glandular epithelium of the endometrium at all stages of the menstrual cycle (206-208). Furthermore, these two CAMs have been detected in menstrual effluent and in endometriosis (206,207). The constitutive expression of both E-cad and P-cad in the endometrium suggests that these two CAMs do not play a role in the highly regulated developmental processes which 44 occurs in this tissue during each menstrual cycle. To date, the cadherin subtypes which are expressed in the stromal compartment of the endometrial epithelium remain to be identified. However, the detection of p-catenin in the human endometrial stroma provides indirect evidence that cadherins are present in this cellular compartment (209). In the placenta, E-cad has been detected in the vil lous cytotrophoblasts but not the extravillous cytotrophoblasts and syncytial trophoblast (188,210). As E-cad is not present in the two trophoblast cell subtypes which form direct interactions with the endometrium, it would seem unlikely that this CAM plays a role in mediating trophoblast-endometrial cell interactions during humari implantation. Furthermore, P-cad, which is believed to be involved in trophoblast-endometrium interactions in the mouse (156), has not been detected in human trophoblast cel ls (164,165). Collectively, these observations suggest that other cadherin subtypes play a role in the cyclic remodeling of the human endometrium and in the complex cellular interactions which occur during human implantation. 1.5.2 Type 2 cadherins in the human endometrium and p lacenta . 45 We have recently determined that the type 2 cadherin subtypes, cad-6 and cad-11, are present in the human endometrium (210,211). Cad-11 was localised to the surface and glandular epithelium at a l l stages of the menstrual cycle examined using immunohistochemistry (210). In contrast, cad-11 expression was first detected in the endometrial stroma during the secretory phase of the menstrual cycle, primarily around the spiral arterioles (the areas of predecidual change). Intense cad-11 immunostaining was observed in the decidua of early pregnancy. Cad-6 mRNA was detected in the human endometrium using reverse transcriptase-polymerase chain reaction (RT-PCR) (211). Cad-6 mRNA transcripts were detected in stromal and glandular epithelial RNA extracts prepared from proliferative and secretory endometrium. In addition, cad-6 appeared to be the predominant cadherin subtype present in the stroma of the proliferative endometrium. Taken together, these observations suggest that these two type 2 cadherins i n the human endometrium may be differentially expressed during the menstrual cycle. In the human placenta, cad-11 was localised to the syncytial trophoblast but not the villous cytotrophoblast cel ls (210). In particular, cad-11 expression was restricted to the apical ruffled 46 border of the syncytial trophoblast and was not detected at the interface between villous cytotrophoblasts and the overlying syncytial trophoblast. Cad-11 expression was also localised to the extravillous cytotrophoblast cell column of the first trimester placenta. Intense cad-11 immunostaining was detected in the cytotrophoblast cells found at the distal end of the column. As cad-11 is expressed in decidua cells and in the two trophoblast cell subtypes (syncytial trophoblast and extravillous cytotrophoblasts) which form intimate interactions with these uterine cells, we speculate that cad-11 plays a central role in human implantation. In particular, cad-11 may mediate trophoblast-decidua cell interactions in a homophilic manner. 1.6 Hypothesis and Rat iona le : The cadherins are a family of CAMs which play a key role in the formation and organisation of tissues during embryonic development and maintain tissue integrity in the adult. In view of these observations, it would seem likely that members of this gene superfamily are involved in the cyclic remodeling processes which occur in the endometrium in preparation for the implanting embryo. The spatiotemporal expression of the classical cadherin subtypes in the 47 human endometrium and placenta suggests that these CAMs do not play a direct role in human implantation. Consequently, we speculated that type 2 cadherin subtypes would be present in the human endometrium. Furthermore, we hypothesised that these CAMs would be spatiotemporally expressed in this tissue and would therefore be l ikely to play a central role in the complex tissue remodeling processes which are involved in preparing the endometrium for the implanting embryo. Our preliminary observations indicated that the two type 2 cadherin subtypes, cad-6 and cad-11, are present in the human endometrium. In these studies, we have localised these two CAMs to the glandular epithelium and stroma of the human endometrium at different stages of the menstrual cycle. In order to determine whether cad-6 and cad-11 are differentially expressed in these two endometrial cellular compartments, Northern blot analysis was performed using total RNA extracts prepared from the epithelium and stroma of the proliferative, mid-secretory and late secretory endometrium. To date, factors capable of regulating type 2 cadherin expression have not been identified. As P4 has been shown to regulate the differentiation of endometrial stroma cells in vitro (65) and classical cadherin levels in the immature mouse uterus (182), we examined the 48 ability of this gonadal steroid to regulate cad-6 and cad-11 expression in isolated endometrial stroma cells. Furthermore, if cad-11 plays a role in human implantation, then alterations in cad-11 expression in the endometrium may adversely effect fecundity. In order to test the hypothesis that a deficiency in cad-11 expression would provide a maternal environment which permits implantation but is subsequently unable to support the developing fetal-placental unit, we have examined the expression of this CAM in endometrial biopsies obtained from women with habitual abortion associated with luteal phase deficiency. Finally, the study of human implantation has been hampered by the lack of a suitable animal model. As a result, we have examined cad-6 and cad-11 expression in the baboon uterus at different stages of the reproductive cycle. These studies will determine whether this non-human primate will provide a model system in which to examine the role of type 2 cadherins in endometrial differentiation and implantation. 49 2. MATERIALS AND METHODS: 2.1 T i s s u e s : Human: Endometrial tissues (n=9) were obtained at the time of hysterectomy from women of reproductive age at at the Dept. Pathology and Laboratory Medicine, University of Pennsylvania. The hysterectomy specimens used in these studies were performed for benign disease. All human tissues obtained in these studies were in accordance with a protocol for the use of human tissue approved by the Committee for Ethical Review of Research involving Human Subjects, University of British Columbia (U.B.C). Informed consent was obtained from al l subjects in these studies. All patients had normal menstrual cycles and had not received hormones for at least three months prior to the collection of tissue. The day of the menstrual cycle was determined by the last menses and confirmed by histological evaluation according to the criteria of Noyes et al. (6). Tissues used in this study were obtained between the mid-proliferative (day 6) and the late secretory phase (day 28) of the menstrual cycle. Endometrial biopsies were obtained from women with habitual abortion associated with luteal phase deficiency. Habitual abortion 50 was defined in these studies as three or more consecutive spontaneous abortions under 20 weeks of gestation, excluding any spontaneous abortions with documented aneuploidy by karyotype analysis. In agreement with Stephenson (61), genetic factors, endocrine factors other than luteal phase deficiency (i.e. thyroid disease and hyperprolactinemia), anatomical factors (septate uterus, intrauterine adhesions or submucousal fibroid) or autoimmune factors (Antiphospholipid Antibody (APA) or Undifferentiated Connective Tissue (UCT) Syndromes) had been diagnosed were excluded from this study. The definition of a luteal phase deficiency used was two endometrial biopsies taken during the mid-secretory phase, of the menstrual cycle with at least three days of maturation delay, as assessed by histological cr i teria (6) and timing of the next menses. A total of 16 women who matched the above-mentioned criteria were recruited from the Recurrent Pregnancy Loss Program, B.C. Women's Hospital and Health Center by Dr. M.D. Stephenson (Dept. Obstetrics and Gynaecology, U.B.C). Dating of the endometrial specimens was performed by Dr. F. Magee (Dept. Pathology, U.B.C). Biopsy specimens from these women were obtained during the secretory phase of the menstrual cycle. 51 Animals: Paraffin embedded sections of baboon (Papio anubis) uterine tissues were kindly provided by Dr. A.T. Fazleabas (Dept. Obstetrics and Gynaecology, University of Illinois, Chicago, II). A l l experimental procedures were approved by the Animal Care Committee of the University of Illinois, Chicago, IL. Uterine tissues were obtained at laparotomy from cycling baboons between the early proliferative (day 3) and late secretory (day 26) phase of the reproductive cycle (n=9). The day of the reproductive cycle was determined by monitoring the pre-ovulatory E2 surge. In addition, baboon uterine tissues were obtained from day 73 of pregnancy (n=3). 2.2 Cell Preparation and Cu l tu re : Epithelial and stroma cells of human secretory endometrium were separated by enzymatic digestion and mechanical dissociation, as described by Sayatswaroop et al. (212). Briefly, the endometrium was minced with a razor blade and subjected to collagenase digestion (0.25%) at 37 °C for 30 min. The stromal cells were isolated from the epithelial glands by passing the cell digest through a nylon sieve (40 urn). Isolated epithelial glands which were retained on the sieve were 52 immediately harvested for Northern blot analysis. The stroma cel ls were collected in a 50 ml tube and purified by layering the supernatant on a Ficoll-Paque gradient and centrifuging the columns at 400 x g for 10 min. Following isolation, the stroma cells were either immediately harvested for Northern blot analysis or plated in Dulbecco's Modified Eagle's medium (DMEM) containing 25 mM glucose, 25 mM Hepes and 50 jug /m l gentamicin and supplemented with 10% heat-inactivated fetal calf serum (FCS). The culture media was replaced 30 min after plating in order to remove epithelial cell contamination. The stroma cells were grown to confluence, washed in phosphate-buffered saline (PBS; pH 7.4), and cultured in DMEM containing charcoal-stripped FCS for a further 24 h. This culture medium was removed and, after the cells had been washed twice in PBS, replaced with fresh DMEM containing charcoal-stripped FCS. P4 (0.1-1 uM), E2 (30 nM) or vehicle (0.1%ethanol) was then added to the culture medium. The cells were harvested for either Northern or Western blot analysis after 24 h of culture in the presence or absence of steroids. 2.3 Immunohis tochemis t rv : 53 Human endometrial tissue specimens obtained from different stages of the menstrual cycle were washed in PBS and fixed in 4% paraformaldehyde/0.1% glutaraldehdye for 2 h at 4 °C. The tissues were washed in PBS in order to remove residual fixative and cryopreserved in a graded series of sucrose (5-15%), prior to embedding the specimens in Tissue Tek O.C.T. Compound (Miles Inc., Elkhart, IN) and being snap-frozen in liquid nitrogen. The blocks of frozen tissue were stored at -70 °C until cryosectioning! Frozen sections (8 um) were prepared from human endometrial tissues obtained during the proliferative, mid-secretory (day 19-23) and late secretory (day 24-28) phases of the menstrual cycle (209,213). The frozen sections were transferred onto glass microscope slides coated with 1% bovine serum albumin (BSA fraction V; Sigma Chem Co., St. Louis, MO), air dried at room temperature (RT) and stored at -70 °C until immunostaining was performed. Immunohistochemistry was performed using either a rabbit polyclonal antibody directed against human cad-6 (Research Genetics Inc., Huntsville, AL) or a mouse monoclonal antibody directed against human cad-11 (designated C11-113E; ICOS Corp., Bothell, WA) (210, 211). Paraffin embedded sections of human and baboon endometrium 54 (10 urn) were cleared in xylene (3 washes, 5 min each), rehydrated in ethanol (3 washes, 5 min each) and washed in running tap water for 5 min. Paraffin-cleared sections and rehydrated frozen sections of endometrium were quenched of endogenous peroxidase activity in a 0.6% hydrogen peroxide (H202)/methanol solution for 20 min at RT. Sequential incubations were performed in a humidified chamber at 37 °C according to the procedures of Cartun and Pederson (214) and included blocking in 10% normal serum (goat or horse serum for the cad-6 and cad-11 antibodies, respectively), primary antiserum for l h , secondary biotinylated antibody for 45 min (goat anti-rabbit IgG and horse anti-mouse IgG for the cad-6 and cad-11 antibodies, respectively; Vector Laboratories, Burlington, CA), streptavidin-biotinylated horseradish peroxidase complex reagent for 30 min (Vector Laboratories, Burlington, CA), and three 5 min washes in PBS. The sections were exposed to chromagen reaction solution (0.035% diaminobenzedine and 0.03% H 20 2) for 5 min, washed in tap water for 5 min, counterstained in Harris haematoxylin and 1% lithium carbonate (LiC0 3), dehydrated, cleared, and mounted. 55 2.4 Northern Blot A n a l y s i s : Total RNA was prepared from the epithelial and stromal components of the human endometrial tissues and the isolated stroma cells by the guanidine thiocyanate phenol-chloroform method of Chbmczynski and Sacchi (215). The RNA species were resolved by electrophoresis in 1% agarose gels containing 3.7% formaldehyde. Approximately 20 u c j of total RNA were loaded per lane. The RNA species were capillary-transferred onto charged nylon membranes (Hybond-N+, Amersham Life Sci., Oakville, ON) in the presence of 2X SSPE (20X SSPE consists of 0.2 M sodium phosphate monobasic, pH 7.4, containing 25 mM EDTA and 3 M NaCI). The Northern blots were incubated in a solution of 3% BSA dissolved in 5X SSPE at 42 °C for 30 min and then transferred to a prehybridisation solution containing 5X SSPE, 50% deionised formamide, 5X Denhardt's solution (5',3', Boulder, CO), 5% dextran sulfate (Pharmacia, Picataway, NJ), 1%SDS, 50 mM sodium phosphate dibasic, and 5 mM sodium phosphate monobasic for 1 h at 42 °C. Heat-denatured sheared salmon sperm DNA (0.2 mg/ml; 5',3', Boulder, CO) and 56 radiolabeled cDNA probes specific for either human cad-11, cad-6 or E-cad were then added to the prehybridisation solution. The cDNA probes were radiolabeled by the random primer method of Feinberg and Vogelstein (216). The blots were incubated with the radiolabeled cDNA probes for 1 6 h at 42 °C. The blots were then washed twice with 2X SSPE at RT, twice with 2X SSPE containing 1%SDS at 55 °C and twice with 0.2X SSPE at RT. The blots were subjected to radioautography in order to detect the hybridisation of the radiolabeled probe to the mRNA species. In order to standardise the amounts of total RNA loaded in each lane, the blots were then reprobed with a radiolabeled synthetic oligonucleotide specific for 18S rRNA according to the protocols of MacCalman et al. (217). The blots were again subjected to radioautography to detect the hybridisation of the oligonucleotide probe to the 18S rRNA. All radioautograms were scanned using an LKB densitometer (LKB, Rockville, MD). The absorbance values obtained with respect to each cadherin mRNA transcript were normalised relative to the 18S rRNA absorbance values. 2.5 Western Blot A n a l y s i s : For Western blot analysis, the stroma cells were incubated in 100 ui cell lysis buffer (Tris-HCl, pH 7.4, containing 0.5% Nonidet P-40, 0.5 mM CaCI2 and 1 mM PMSF) at 4 °C for 30 min on a rocking platform. The cell lysates were centrifuged at 10, 000 x g for 20 min and the supernatants collected for use in the Western blot analysis. Protein concentrations in the supernatants were spectrophoreticaly assessed using a Bio-Rad Detergent Compatible Protein Assay kit (Bio-Rad Life Sci., Mississauga, ON). Standardised aliquots of protein were then taken and subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions (5% p-mercaptoethanol), as described by Laemmli (218). The stacking gels contained 5% acrylamide and the separating gels were composed of 7.5% acrylamide. The proteins were electrophoretically transferred from the gels onto nitrocellulose paper (Hybond ECL, Amersham Life Sci., Oakville, ON) according to the procedures of Towbin et al. (219). The nitrocellulose blots were probed with a rabbit polyclonal antibody directed against human cad-6 (Research Genetics Inc., Huntsville, AL) or a mouse monoclonal antibody directed against human cad-11 58 (designated C11-113H; ICOS Corp., Bothell, WA). The Amersham Enhanced Chemiluminescence (ECL) system was used to detect antibody bound to antigen. 2.6 Stat is t ica l A n a l y s i s : The results derived from the Northern and Western blots are presented as the mean relative absorbance (± SE) for three independent experiments. Statistical differences between separate time points and treatments were assessed by the analysis of variance (ANOVA). Differences were considered to be significant for p<0.05. Significant differences between the means were determined using the least significant test. 59 3. RESULTS 3.1 Localisation of cadherin-6 and cadherin-11 in the human endometrium during the menstrual c y c l e . Cad-6 was detected in the glandular epithelium and stroma of the human endometrium during the proliferative phase of the menstrual cycle (Fig. 2). The intensity of cad-6 immunostaining was high in the glandular epithelium during the proliferative phase but declined as the cycle entered the secretory phase. Similarly, the intensity of cad-6 immunostaining was high in the endometrial stroma during the proliferative and mid-secretory phases but appeared to decline as the stroma cells underwent decidualisation during the late secretory phase of the menstrual cycle. Cad-11 was detected in the glandular epithelium at all stages of the menstrual cycle (Fig. 2). The intensity of immunostaining appeared to remain constant throughout the menstrual cycle. In the stroma, cad-11 was first detected in the predecidualised cells surrounding the spiral arterioles during the mid-secretory phase. The expression of cad-11 in the endometrial stromal cells appeared to be concomitant with the loss of cad-6 expression (Fig. 2). Figure 2: Immune-localisation of cad-6 and cad-11 in the human endometrium. Cad-11 was detected in the glandular epithelium of the proliferative, mid-secretory and late secretory endometrium (panels A, C and E respectively). Intense cad-11 immunostaining was only detected in the stroma during the late secretory phase (panel E). In contrast, cad-6 was detected in the glandular epithelium and stroma during the proliferative phase (panel B). There was a marked decrease in the intensity of cad-6 immunostaining during the mid- and late secretory phases of the menstrual cycle (panel D and F, respectively). The loss of cad-6 expression in the endometrial stroma (panel F) was concomitant with the appearance of cad-11 in this cellular compartment (panel E). Negative controls in which the cad-11 and cad-6 antisera were omitted are shown in panels G and H, respectively. 61 3.2 Expression of the cadherin subtypes in the g landu lar epithelium and stroma of the human endometrium during the menstrual c v c l e . In order to determine whether the type 2 cadherin subtypes present in the human endometrium were being differentially expressed, we examined cad-11 and cad-6 mRNA levels in glandular epithelial and stromal cell total RNA extracts prepared from the proliferative and secretory endometrium using Northern blot analysis. In addition, to confirm that the stromal cell preparations were not contaminated with epithelial cells, we also examined E-cad mRNA levels, a cadherin subtype previously localised to the glandular and surface epithelium of the human endometrium (206-209). A single E-cad mRNA transcript of 5.6 kb was detected in total" RNA extracts prepared from the glandular epithelium at all stages of the menstrual cycle (Fig. 3). We failed to detect E-cad mRNA transcripts in any of the endometrial stroma cell extracts. A single cad-6 mRNA transcript of 4.1 kb was detected in total RNA extracts prepared from glandular epithelium and endometrial stroma at all stages of the menstrual cycle (Fig. 3). There was a 63 significant decline in epithelial and stromal cad-6 mRNA levels as the cycle entered the secretory phase. The levels of the cad-6 mRNA transcript continued to decline during the late secretory phase of the menstrual cycle. A single cad-11 mRNA transcript of 4.4 kb was detected in the glandular epithelium at all stages of the menstrual cycle (Fig. 3). We failed to detect cad-11 mRNA transcripts in the endometrial stroma until the mid-secretory phase of the menstrual cycle. Stromal cad-11 mRNA levels continued to increase as the cycle entered the late secretory phase. 64 Figure 3: Radioautograms of Northern blots containing total RNA extracted from the stroma (lanes a-c) or glandular epithelium (lanes d-f) of the human endometrium during the late proliferative (lanes a and d), mid-secretory (lanes b and e) and late secretory phase (lanes c and f) of the menstrual cycle. The blot was probed for cad-11, cad-6, E-cad, and 18S rRNA. The positions of the 28S and 18S rRNA species are shown on the left side of the upper panel. The radioautograms were scanned using a laser densitometer and the values for the cad-11, cad-6 and E-cad mRNA transcripts were then normalised relative to the absorbance value obtained for the 18S rRNA. The results derived from this analysis, as well as those from two other studies (radioautograms not shown), were standardised relative to the cadherin mRNA levels in the stroma or glandular epithelium of the proliferative endometrium and are represented (mean ± SE; n=3) in the bar graphs (*p<0.05). 65 a b c d e f 2 8 S -cad-11 18S-2 8 S -cad -6 18S-\ B (' D E I Stroma Glandular Epithelium A H (' D E Stroma Glandular Epithelium A ll (' I) E I Stroma Glandular Epithelium 3.3 Differential regulation of cadherin-6 and cadherin-1 1 in cultured endometrial stroma cells bv the gonadal s t e r o i d , progesterone. As a first step in identifying the factors capable of regulating the type 2 cadherins present in the human endometrium, we examined the effects of P4 on cad-6 and cad-11 mRNA levels in isolated endometrial stroma cells. P4 increased cad-11 mRNA levels in a dose-dependent manner (Fig. 4). The increase in cad-11 mRNA levels was concomitant with a decrease in cad-6 mRNA levels. Western blot analysis using extracts prepared from stroma cel ls treated with P4 and either the mouse monoclonal antibody directed against human cad-11 (C11-113H) or the rabbit polyclonal antibody directed against human cad-6 revealed a single cad-11 protein species (Mr 125 kDa) and a single cad-6 protein species (Mr 120 kDa), respectively (Fig. 5). In agreement with the Northern blot analysis, the P4-induced increase in cad-11 levels was concomitant with a decrease in cad-6 expression in these cells. Figure 4: Radioautograms of Northern blots containing total RNA extracted from isolated endometrial stroma cells treated with vehicle (lane A), 0.1 uM P4 (lane B), 0.5 P4 (lane C), 1 uM P4 (lane D), or 30 nM E2 (lane E). The blots were probed for cad-11 (upper panel), cad-6 (middle panel) or 18S rRNA (lower panel). The positions of the 28S and 18S rRNA species are shown on the left side of the upper panel. The radioautograms were scanned using a laser densitometer and the values for the cad-11 and cad-6 mRNA transcripts were then normalised relative to the absorbance value obtained for the 18S rRNA. The results derived from this analysis, as well as those from two other studies (radioautograms not shown), were standardised relative to the vehicle control and are represented (mean ± SE; n=3) in the bar graphs (*p<0.05). 68 A B C D E Figure 5: Western blot analysis of cad-11 (upper panel) or cad-6 (lower panel) in isolated endometrial stroma ceils. Thirty \ig of protein extracted from cells cultured in the presence of vehicle alone (lane A), 0.5 uM P4 (lane B) or 1 uM P4 (lane C) were loaded in each lane. Western blot analysis was performed using a mouse monoclonal antibody directed against human cad-11 or a rabbit polyclonal antibody directed against cad-6. The Amersham ECL system was used to detect antibody bound to antigen. The radioautograms were scanned using an LKB laser densitometer. The results derived from this analysis, as well as from two other studies (radioautograms hot shown) were standardised relative to the vehicle control and are represented (mean ± SE; n=3) in the bar graphs (*p<0,05). 70 A B C kDa 200-CAD-11 6 9 -200-Treatment 3.4 Cadherin-11 expression in the endometrium of women with habitual abortion associated with luteal phase de f i c i ency . In order to determine whether endometrial cad-11 expression is altered in women with reproductive health problems, we examined the expression of this CAM in endometrial biopsies obtained from women with habitual abortion associated with luteal phase deficiency. Cad-11 was not detected in the luminal or glandular epithelium in this cohort of women (Fig. 6). We also failed to detect cad-11 in the stroma of the secretory endometrium in these women. In contrast, cad-1 T immunostaining was readily detectable in the epithelial and stromal compartments of secretory phase endometrial biopsies obtained from fertile controls (Fig. 6). 72 Figure 6: Immune-localisation of cad-11 in the endometrium of women with habitual abortion associated with luteal phase deficiency during the secretory phase of the menstrual cycle (panel A). A positive control of secretory endometrium obtained from fertile women immunostained for cad-11 is shown in panel B (le=luminal epithelium, ge=glandular epithelium, st=stroma). 73 74 3.5 Localisation of cadherin-6 and cadherin-11 in the baboon (Paoio anubis) u terus. In order to determine whether the non-human primate is a suitable model for examining the role of type 2 cadherins in endometrial differentiation and implantation, we localised cad-6 and cad-11 in the baboon uterus at different stages of the reproductive cycle. Cad-6 was detected in the glandular epithelium and stroma of the proliferative endometrium (Fig. 7). The intensity of cad-6 immunostaining appeared to decline in both of these cellular compartments as the cycle entered the secretory phase. Low levels of cad-6 immunostaining were present in the glandular epithelium and stroma during the late secretory phase of the reproductive cycle. Cad-11 immunostaining was detected in the glandular epithelium of the baboon endometrium at all stages of the reproductive cycle examined (Fig. 8). The levels of cad-11 in this cellular compartment appeared to remain constant throughout the reproductive cycle. In contrast, we failed to detect cad-11 expression in the endometrial stroma during either the proliferative or mid-secretory phase. Cad-11 expression was first detected in the stroma in regions surrounding the 75 spiral arteries (the areas of early decidualisation) during the late secretory phase of the reproductive cycle. Maximum cad-11 immunostaining in the baboon uterus was observed in the decidua of early pregnancy (Fig. 9). In contrast, we failed to detect cad-11 in the cells which constitute the spiral arteries present in this tissue layer. We detected immunostaining for cad-6 and cad-11 in the smooth muscle cells of the baboon myometrium at all stages of the reproductive cycle examined (Fig. 10). The intensity of immunostaining for these two CAMs appeared to remain constant throughout the reproductive cycle. In contrast, we did not detect cad-6 or cad-11 immunostaining in the interstitial stroma cells of the myometrium. 76 Figure 7: Immune-localisation of cad-6 in the baboon endometrium. Cad-6 was detected in the glandular epithelium and stroma during the proliferative phase of the reproductive cycle (Panel A). In the mid-secretory endometrium, reduced levels of cad-6 immunostaining were present in the glandular epithelium and the stromal cells (Panel B). The expression of cad-6 continued to decline in both of these cellular compartments during the late secretory phase of the reproductive cycle (Panel C). Panel D shows a negative control in which primary antiserum was omitted. 77 Figure 8: Immunolocalisation of cad-11 in the baboon endometrium. Cad-11 was detected in the glandular epithelium during the proliferative, mid-secretory and late secretory phase of the reproductive cycle (Panels A, B and C, respectively). Cad-11 immunostaining was first detected in the endometrial stroma in the cells surrounding the spiral arteries during the late secretory phase (Panel C). Primary antiserum was omitted as a negative control (Panel D). 79 Figure 9: Immune-localisation of cad-11 in the early pregnancy decidua of the baboon. Intense immunostaining for cad-11 was detected in the decidua cells of the pregnant baboon uterus (Panel A and B). We failed to detect cad-11 expression in the cells which constitute the spiral arteries in this tissue layer. Panel C shows a negative control in which primary antiserum was omitted. 81 Figure 10: Immune-localisation of cad-6 and cad-11 in the baboon myometrium. Cad-6 was detected in the smooth muscle cells of the myometrium, but not the interstitial stroma cells, during the proliferative and secretory phases of the reproductive cycle (Panels A and C, respectively). Cad-11 immunostaining was detected in the myometrial smooth muscle cells during the proliferative and secretory phases of the reproductive cycle (Panel B and D, respectively). Negative controls in which the cad-11 and cad-6 antisera were omitted are shown in panels E and F, respectively. 83 4. DISCUSSION: Previous studies have failed to identify all of the cadherin subtypes present in the human endometrium. Van der Linden et al. (205,206) detected E-cad and P-cad in the luminal and glandular epithelium but not in the stroma of the human endometrium at all stages of the menstrual cycle. Our findings indicate that the type 2 cadherin subtypes, known as cad-6 and cad-11, are spatiotemporally expressed in the glandular epithelium and stroma of the human endometrium during the menstrual cycle. The expression of cad-6 and cad-11 is differentially regulated" in the endometrial stroma (Fig. 11). Cad-6 is expressed in the endometrial stroma during the proliferative phase of the menstrual cycle. There is a marked reduction in the levels of cad-6 as the stroma undergoes decidualisation during the secretory phase. The loss of cad-6 expression is concomitant with an increase in cad-11 expression. The differential regulation of cad-6 and cad-11 has been previously observed during the terminal differentiation of other human cell types. 8 5 Figure 11: Correlation of the spatiotemporal expression of cad-6 and cad-11 in the human endometrium with the cyclic remodeling processes described by Noyes et al. (6). During the proliferative phase of the menstrual cycle, the stroma and glandular epithelium are mitotically active. Cad-6 and cad-11 are present in the glandular epithelium during this stage of the menstrual cycle. However, only cad-6 i s detected in the stroma of the proliferative endometrium. As the cycle enters the secretory phase, mitotic activity in both cellular compartments is reduced. During the secretory phase, the glands become secretory and the stroma undergoes decidualisation. These developmental processes are correlated with a decline in cad-6 expression in both cellular components and a marked increase in cad-11 expression in the endometrial stroma. 86 So glandular epithelium • H M M . proliferation secretion cadherins PH cad-6 H cad-11 stroma M M M M H proliferation decidualisation cadherins cad-6 PH cad-11 For example, we have determined that cad-6 mRNA levels decrease and the levels of the cad-11 mRNA transcript increase in human granulosa cells undergoing spontaneous leutinisation in culture (202). Similarly, cad-6 is present in undifferentiated myoblast cells but not myotubes (220). There is a marked increase in cad-11 mRNA levels following the formation of the terminally differentiated myotube in culture. Taken together, these observations suggest that cad-6 expression may be restricted to progenitor cells in the adult human and that the terminal differentiation of these cells is associated with a marked increase in cad-11 expression. The role of cad-11 in cellular differentiation has not yet been determined. The expression of cad-6 and cad-11 does not appear to be complementary in the glandular epithelium of the human endometrium (Fig. 11). Cad-6 is expressed during the proliferative phase, but is down-regulated during the secretory phase of the menstrual cycle. I n contrast, the levels of cad-11 and E-cad in this cellular compartment remain constant throughout the menstrual cycle. These observations suggest that the glandular epithelium of the human endometrium expresses multiple cadherin subtypes at any given time during the menstrual cycle. Multiple cadherin subtypes have been detected in 88 several other cell types including human granulosa cel ls (211), human fibroblasts (221), renal carcinoma cel ls (201), and mouse thymocytes (222). The biological relevance of these complex expression patterns remains to be elucidated. The presence of E-cad and cad-11 in the glandular epithelium is the first demonstration that these two CAMs can be coordinately expressed in a single cell type. For example, Shimazui et al. (201) failed to detect coordinate expression of E-cad and cad-11 in 20 renal carcinoma cell lines, despite observing the coexpression of other type 1 and type 2 cadherin subtypes. In addition, murine prostate cancer cell lines which express E-cad do not contain mRNA transcripts for cad-11, whereas the loss of E-cad in these cells is frequently accompanied with an increase in the levels of cad-11 mRNA (223). Finally, we have demonstrated that the terminal differentiation of trophoblast cells isolated from term placenta is associated with a switch from E-cad to cad-11 expression (210). The mechanism(s) which are involved in the cell-specific regulation of these two cadherin subtypes remains to be elucidated. The accumulation of cad-11 in the endometrial stroma correlates with the early events of implantation in the human (4). As we have 89 previously localised cad-11 to the syncytial trophoblast and extravillous cytotrophoblast cell column of the human placenta (210), the two cell types which form intimate interactions with the decidualised cells of the endometrium, it is tempting to speculate that cad-11 mediates trophoblast-decidual cell interactions in a homophilic manner. This cellular interaction may anchor the trophoblast cells to the decidua and arrest their invasion. This hypothesis is consistent with the findings of Shibata et al. (224), who reported that cad-11 may be involved in arresting tumour cell invasion by mediating carcinoma cell-stroma cell interactions. In addition, Bussemakers et al. (225) recently detected cad-11 mRNA transcripts in several prostate cancer cell lines. The authors speculated that the expression of cad-11 in prostate carcinomas may play a role in bone metastasis by mediating carcinoma cell-osteoblast cell interactions in a homophilic manner. To date, the ability of cad-11 to modulate cellular invasion has not been determined. We have previously demonstrated that the gonadal steroids, E2 and P4, are key regulators of the classical cadherins in the reproductive tracts of the immature mouse (182,183,186,187). For example, both of these gonadal steroids are capable of regulating E-cad 9 0 mRNA levels in the immature mouse uterus in vivo (182). These studies also indicated that E2 and P4 regulated E-cad mRNA levels by different mechanisms, as only actinomycin D (an inhibitor of RNA transcription) and not cyclohexamide (an inhibitor of protein synthesis), was able to prevent the P4-induced increase in E-cad mRNA. In contrast, both inhibitors prevented the increase in E-cad mRNA transcripts in response to E2. In these studies, we have determined that P4 is also a key regulator of the type 2 cadherin subtypes present in the stroma of the human endometrium. However, E2 does not appear to be capable of regulating either cad-6 or cad-11 mRNA levels in cultured endometrial stroma cells. These observations suggest that E2 and P4 likely work through different molecular mechanisms to regulate the expression of the endometrial cadherin subtypes. Collectively, these studies provide further support for our working hypothesis that gonadal steroids mediate the cyclic remodeling of the female reproductive tract, at least in part, through their ability to regulate the expression of different cadherin subtypes. In the human, both ovarian steroids, as well as other factors such as prostaglandins, gonadotropins and various growth factors, are required for decidualisation of the endometrial stroma (45,48,51,65). 91 The actions of these multiple factors in the endometrium are likely to be mediated through their ability to regulate the expression of several molecular components of decidualisation in vivo. For example, Grossinsky et al. (226) recently reported that several growth factors are capable of modulating integrin subunit expression in human endometrial stroma cells. In view of these observations, it is tempting to speculate that the complex combination(s) of regulatory factors which are likely to be involved in the process of decidualisation are required for the induction of maximum levels of cad-11 in isolated endometrial stroma cells. The expression of cad-11 in the glandular epithelium is not coordinated with the cycle-specific changes observed for this CAM in the endometrial stroma. > For example, cad-11 is expressed in the glandular epithelium during the proliferative phase of the menstrual cycle when E2 is the predominant ovarian steroid. In addition, the levels of this CAM do not increase in the epithelial compartment during the P4-dominated secretory phase. These observations indicate that multiple factors regulate epithelial cad-11 expression and strengthen our hypothesis that several factors are likely to be involved in regulating type 2 cadherin expression in the human endometrium. 92 We did not examine the factors capable of regulating cad-6 and cad-11 expression in the glandular epithelial cells isolated from the human endometrium as these cells fail to maintain many of their in vivo characteristics. For example, the polarised epithelial phenotype is not maintained (227) and there is a marked reduction in the number of steroid receptors expressed by these cells in vitro (228). Although three-dimensional culture systems of human endometrial epithelial cells using the reconstituted basement membrane matrix, Matrigel, have been shown to display many of the characteristics of epithelial cells in vivo and prolong the ability of these cells to respond to steroids in culture (229,230), the presence of endogenous growth factors, such as bioactive TGFp-l, in Matrigel may confound the results of these experiments (231). Recent studies indicate that endometrial adenocarcinoma cell lines may provide a suitable alternative for studying the cell biology of glandular epithelial cells (232,233). For example, the Ishikawa cell line maintains hormonally-inducible E2 and P4 receptors in culture (232) and exhibits some of same patterns of integrin expression described in vivo (233). However, this cell line does not express the a4 and pi integrin subunits, two putative markers of endometrial receptivity in the human (233). Taken together, these 9 3 observations indicate that endometrial cell cultures are not ideal model systems for identifying the factors capable of regulating type 2 cadherins in the human endometrium. Our observations in the human endometrium suggest that the regulation of the type 2 cadherins in this tissue layer is likely to be complex. In order to address this issue, the untranslated regions of the genes which encode for cad-6 and cad-11 will have to be cloned and characterised. To date, the only cadherin promoter regions which have been partially characterised are the mouse and human E-cad gene (234,235). These studies have identified three putative P4 receptor binding sites (234), strengthening our hypothesis that gonadal steroids are key regulators of cadherin expression in mammalian tissues. Cloning of the cad-6 and cad-11 untranslated regions is likely to be a difficult task, as the murine E-cad and N-cad genes are 40 kb (234) and 200 kb (236) in length, respectively, with only 5 kb comprising exons in both classical cadherin genes. The spatiotemporal expression of cad-6 and cad-11 in the endometrium of the baboon is consistent with the expression patterns observed for these two CAMs in the human. Recently, several molecular markers which are spatiotemporally expressed in the human 94 endometrium have been detected in the baboon uterus. For example, the integrin subunits a l , a4 and p3 in the glandular epithelium of the baboon endometrium are only detected during the secretory phase of the reproductive cycle, corresponding with the cycle-specific expression pattern of these CAMs in the human endometrium (237). In addition, the expression of MUC1 in the luminal epithelium is up-regulated during the peri-implantation period in both the baboon and the human endometrium (238). These observations suggest that several characteristics of the human endometrium are conserved in the baboon at the molecular and cellular level. As a result, the baboon may serve as a suitable model for examining the molecular events surrounding embryo implantation in vivo. Although we detected cad-11 in the baboon endometrial stroma during the secretory phase of the reproductive cycle, immunostaining for this CAM was restricted to regions surrounding the spiral arteries. In contrast, decidua obtained from early pregnancy in these animals exhibited a more extensive cad-11 staining pattern. The process of decidualisation in the human and the baboon differs with respect to timing and the factors which are necessary to regulate this cellular process (67,68). In particular, terminal differentiation of the 95 endometrial stroma cells into decidua cells in the baboon endometrium requires the presence of an implanting embryo. The temporal delay in baboon decidualisation may allow for a more detailed analysis of the sequence of molecular events which occur during this cellular differentiation process (68). We also detected cad-6 and cad-11 expression in the smooth muscle cells of the myometrium in the baboon uterus. Although the myometrium is exposed to a similar cyclic hormonal milieu as the endometrium and is responsive to gonadal steroids (239), the levels of these two CAMs did not appear to change in the myometrium during the reproductive cycle. These findings lend further support to our hypothesis that multiple factors act in a cell-specific fashion to regulate the expression of cad-6 and cad-11 in the uterus. Previous studies have demonstrated the presence of the classical cadherin subtype, N-cad, in the human myometrium (240). It remains to be determined whether the human myometrium also expresses the two type 2 cadherin subtypes, cad-6 and cad-11. Cad-6 and cad-11 were not localised to the cell membranes of the glandular epithelial and stromal cells of the human and baboon endometrium using immunohistochemistry. The significance of the 96 diffuse staining pattern observed throughout the cytoplasm of these endometrial cells remains unclear. Previous studies have failed to localise type 2 cadherins to areas of cell contact in the human placenta (210,211,241) and endometrium (210,211). In addition, prominent cytoplasmic staining of different classical cadherin subtypes has been described in vivo and in vitro using several different, w e l l -characterised antibodies (240^246). For example, diffuse cytoplasmic staining has been observed in tissue sections prepared from the human endometrium and myometrium (N-cad; 240), placenta (N-cad; 241), prostate (P-cad; 242), ovary (N-cad and E-cad; 243) lung (N-cad and E-cad; 244,245) and baboon ovary (E-cad; 246). Extrajunctional disribution of the classical cadherins has also been detected in several cell culture systems (126,247-249). In particular, E-cad has been detected in the cytoplasm of MDCK cells; an in vitro model which i s used extensively to study epithelial cell polarity (177). Recently, Peralta Soler et al. (242) reported that the cellular distribution of P -cad is associated with cellular proliferation of prostatic carcinoma cells. However, it remains unclear whether the cytoplasmic staining pattern obtained for the classical and type 2 cadherins has biological significance or whether it is the result of technical artifacts arising 97 from the processing and fixation of the tissues, or non-specific binding of the cadherin antibodies to other cytoplasmic proteins. the cellular distribution of the cadherins can be modified by the use of non-ionic detergents, such as Triton X-100. For example, the permeabilisation of MDCK cell membranes results in a more restricted distribution of E-cad at regions of cell-cell contact (247,249). In addition, a significant proportion of E-cad protein is recovered in the Triton X-100 soluble fraction obtained from MDCK cells (247,250,251). It remains to be determined whether the Triton X-100 soluble and insoluble fractions of E-cad protein represent functionally distinct cellular pools of this CAM or whether they reflect different stages of E-cad containing complex assembly and/or processing. The specificity of the antibodies directed against the human cadherin subtypes have been determined by a combination of Western blot analysis, immunoprecipitation, and/or immunohistochemistry (252). The monoclonal antibody directed against human cad-11 which was used in these studies has been shown to recognise a single protein species of 125 kDa using Western blots containing total protein extracts prepared from choriocarcinoma cells (210) and isolated endometrial stromal cells. In addition, the immunostaining pattern 98 observed in the endometrial tissue sections has been repeated using another monoclonal antibody directed against cad-11 (kind gift from Dr. Karen A. Knudsen, Lankenau Medical Centre, Philadelphia, PA), suggesting that the antibody used in these studies is specific for cad-11. Similarly, the cad-6 polyclonal antibody recognises a single protein species of 120 kDa using Western blots containing total protein from human endometrial cells and the rodent and human kidney. These cellular extracts contain multiple cadherin subtypes which are not recognised by the polyclonal antibody used in these studies. In addition, the differential localisation of the two cadherin subtypes in the human and baboon endometrium and the differences in the levels and moblilities of the cad-6 and cad-11 proteins species observed using Western blots containing endometrial stroma cells cultured in the presence or absence of P4 suggest that both antibodies are specif ic for each respective cadherin subtype. Further evidence to suggest that the antibodies directed against cad-6 and cad-11 are specific for the respective cadherin subtypes is obtained from the Northern blot analyses performed using total RNA extracted from endometrial tissue specimens and isolated endometrial stroma cells. The mRNA levels of the two endometrial cadherin 99 subtypes reflects the protein levels observed in these cellular preparation. A direct correlation between cadherin expression and mRNA levels has previously been reported for N-cad (253), E-cad (189), cad-6 (199) and cad-11 (210). The use of the histological criteria of Noyes et al. (6) for dating of the endometrium is currently considered the most reliable method for diagnosing aberrant luteal phase maturation of the glandular epithelium and/or endometrial stroma (3,254). Significant interobserver and intraobserver variability in endometrial dating using morphological parameters suggests that other methods for determining luteal phase maturation of the endometrium are required (255,256). the use of molecular markers as immunohistochemical tools in order to accurately define endometrial function may serve as a useful alternative to these classical diagnostic criteria. Proteins whose expression is associated with the molecular cascade of events surrounding implantation are potential candidates for such immunohistochemical markers of endometrial differentiation. Aberrant luteal phase maturation of the endometrium is believed to be a major cause of infertility in women (3). Previous studies have demonstrated that the spatiotemporal expression of several integrin 100 subunits in the endometrium is highly regulated during this phase of the menstrual cycle (99-102). As the appearance of these integrin subunits corresponds with the putative window of implantation in the human, it has been postulated that these CAMs play a central role in implantation. Although the role of these integrin subunits in implantation has not been determined, the disruption of this expression pattern has been associated with primary infert i l i ty (103). In view of these observations, it is tempting to speculate that the switch in cad-6 to cad-11 expression in the endometrial stroma may also act as a marker for the luteal phase maturation of the human endometrium. Failure to regulate cad-11 expression in the endometrial stroma may therefore have a profound effect on fecundity. We failed to detect cad-11 expression in the secretory endometrium of women with habitual abortion associated with luteal phase deficiency in these studies. These findings are in direct contrast with our observations using endometrial biopsies obtained from fert i le women. For example, cad-11 expression was detected in the glandular epithelium of fertile women at all stages of the menstrual cycle. In contrast, we did not detect cad-11 immunostaining in the glandular epithelium of women diagnosed with habitual abortion, using 101 endometrial biopsies obtained from the secretory phase of the menstrual cycle. Taken together, these observations suggest that an endometrium that fails to express cad-11 in either the epithelial or stromal compartment is initially receptive to the implanting embryo but is not able to maintain the developing fetal-placental unit as pregnancy progresses. The functional implications of these aberrant expression patterns in the endometrium remain to be determined. However, these observations indicate that molecular markers of endometrial receptivity may not prove useful for assessing the likelihood of pregnancy in this cohort of women with habitual abortion associated with luteal phase deficiency. Instead, markers for a 'competent endometrium' which allows for both implantation and maintenance of the embryo may be more effective in these cl in ical circumstances. 102 5. CONCLUSIONS: In conclusion, these studies demonstrate that the two type 2 cadherin subtypes, cad-6 and cad-11, are spatiotemporally expressed in the endometrium during the menstrual cycle. P4 is capable of differentially regulating the expression of these two cadherin subtypes in isolated human endometrial stroma cells. Collectively, these observations suggest that cad-6 and cad-11 may play a central role in the cellular remodeling processes which occur in the human endometrium during the menstrual cycle. In addition, we failed to detect cad-11 expression in the endometrium of women with habitual abortion diagnosed with luteal phase deficiency. These findings indicate that cad-11 may prove to be a reliable marker for the diagnosis of habitual abortion associated with luteal phase deficiency. 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