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In Situ and in Vitro Immunolocalization of Oviductin, an Oviduc-Specific Glycoprotein, in the Hamster Uterine Epithelial Cells

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In Situ and in Vitro Immunolocalization of Oviductin, an Oviduc-Specific Glycoprotein, in the Hamster Uterine Epithelial Cells IN SITU AND IN VITRO IMMUNOLOCALIZATION OF OVIDUCTIN BINDING SITES ON HAMSTER UTERINE EPITHELIAL CELLS AND DETECTION OF A HAMSTER OVIDUCTIN HOMOLOGUE IN THE FEMALE RAT REPRODUCTIVE TRACT by YING ZHENG A thesis submitted to the Department of Anatomy and Cell Biology in conformity with the requirements for the degree of Master of Science Queen’s University Kingston, Ontario, Canada February 2008 Copyright © Ying Zheng, 2008 ABSTRACT Oviductin is an oviduct-specific and high-molecular-weight glycoprotein that has been suggested to play important roles in the early events of reproduction. The present study was undertaken to localize the oviductin binding sites in the uterine epithelial cells of the golden hamster (Mesocricetus auratus) both in situ and in vitro, and to detect a hamster oviductin homologue in the female rat reproductive tract. Immunohistochemical localization of oviductin in the hamster uterus revealed certain uterine epithelial cells reactive to the monoclonal anti-hamster oviductin antibody. In order to study the interaction between hamster oviductin and the endometrium in vitro, a method for culturing primary hamster uterine epithelial cells has been established and optimized. Study with confocal microscopy of the cell culture system showed a labeling pattern similar to what was observed using immunohistochemistry. Pre-embedding immunolabeling of cultured uterine epithelial cells also showed gold particles associated with the plasma membrane and microvilli. These results demonstrated that hamster oviductin can bind to the plasma membrane of certain hamster uterine epithelial cells, suggesting the presence of a putative oviductin receptor on the uterine epithelial cell surface. In the second part of the present study, using the monoclonal anti-hamster oviductin antibody that cross-reacts with the rat tissue, we have been able to detect an oviduct-specific glycoprotein, with a molecular weight of 180~300kDa, in the female rat reproductive tract. Immunohistochemical labeling of the female rat reproductive tract revealed a strong immunolabeling in the non-ciliated oviductal epithelial cells and a faint immunoreaction on the cell surface of some uterine epithelial cells. Ultrastructurally, ii immunogold labeling was restricted to the secretory granules, Golgi apparatus, and microvilli of the non-ciliated secretory cells of the oviduct. In the uterus, immunogold labeling was observed on the cell surface of some uterine epithelial cells. Furthermore, electron micrographs of ovulated oocytes showed an intense immunolabeling for rat oviductin within the perivitelline space surrounding the ovulated oocytes. The findings of the present study demonstrated that oviductin is present in the rat oviduct and uterus, and it appears that, in the rat, oviductin is secreted by the non-ciliated secretory cells of the oviduct. iii Dedicated to my parents, parents in law, and husband Thank you for your love and constant support that led me all the way to here. I love you. iv ACKNOWLEDGEMENTS First and foremost, I would like to sincerely thank my graduate supervisor, Dr. Frederick.W.K. Kan for his support, patience, guidance and encouragement in the past years. Without his support and help, it would be impossible for me to finish this thesis. Studying at Queen’s and working in his laboratory were unforgettable and valuable experience in my life. I also want to express many thanks to my co-workers in the lab. Changnian Shi was our technician who patiently taught me useful techniques and gave me good suggestions throughout my project. Bo Kong and Laurelle Saccary are my labmates who have made my overseas studying so fulfilling. I would like to thank all of you for such enjoyable memories that we shared. I’d like to extend a special thank to Mr. John Dacosta and Dr. Xiaohu Yan for their wonderful technical assistance. Over the past several years, I received tremendous support and help from members within and outside the department. I want to say thank you to Dr. Kan, Dr. Reifel, Dr. Oko, and Dr.Mackenzie for their wonderful lectures and lessons. I want to show my appreciation to Dr. Oko, Dr. Graham and Dr. Leda Raptis for letting me use their facilities. Thanks to Dr. Virginia Walker and Dr. Inka Brockhausen for kindly providing me with cell lines. Wei Xu, Yang Yu, Judy and Zheng Qin from Dr. Oko’s lab were so friendly that I can easily talk to and ask questions. A special thank is given to my good friend, Jun Liu, Ph.D candidate in the Department of Biology. Not only did I learn many molecular biology techniques from him, but also I learned a great deal of scientific faith from him. In a word, without their help, it would have been impossible for me to finish my project. To the departmental technical and administrate staffs, including Mr. Rick Hunt, Mr. Donaldson Earl, Mrs. Anita Lister, Mrs. Marilyn McAuley, Ms. Ita A McConnell and others, thank you for your kind assistance. To all my fellow graduate students and my friends, because of you, I have a wonderful and unforgettable experience studying at Queen’s University, Canada. Thank you indeed! Finally, but not the least, I am extremely thankful to my beloved families, especially to my husband Zhuoran Cai, my parents and parents in law. Without their true love and unconditional support, I would not have had the current achievement. v TABLE OF CONTENTS Page ABSTRACT ………………..……………………………………….……...………... ii DEDICATION ....……………….………………………………………..….………. iv ACKNOWLEDGEMENTS ….…………………………………………..………… v TABLE OF CONTENTS …..……………………………………………..………… vi LIST OF FIGURES AND ILLUSTRATIONS ….……..…………………..……… x LIST OF TABLES ….…………………………………………………..…………… xii LIST OF ABBREVIATIONS …..………………………………………..…..……... xiii CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW …….……... 1 1 OVERVIEW …....……………………………………………………..………. 2 2 LITERATURE REVIEW ……………………………………………..….….. 4 2.1 The Female Reproductive Tract ……………………………..…......……... 4 2.2 Uterus ……………………………….………..…………….…..….…….... 5 2.2.1 Anatomical Structure of the Uterus …………………………...….. 5 2.2.2 Histological Features of the Uterus ………….…………..…...…... 6 2.2.3 Hormonal Regulation in the Uterus …………….…………..……. 7 2.2.4 Cyclic Changes in the Endometrium of the Uterus …...……….…. 8 2.2.5 Implantation in the Uterus …...………..………………..……..….. 12 2.2.6 Uterine Epithelial Cell Culture System ……………..………..…... 13 2.3 Oviduct ……………………………………………...…………….……… 16 2.3.1 Anatomical Structure of the Oviduct ……………………....……... 16 2.3.2 Histological Features of the Oviduct ………………..……..……... 17 2.4 Oviductal Fluid …………………………………………………..…..…… 20 2.5 General Review of Oviductin ………………………….….………...……. 21 2.6 Hamster Oviductin ……………………………………..………..………... 26 2.6.1 Ontogeny of Hamster Oviductin ………………………..…...……. 26 2.6.2 Biochemical Characterization of Hamster Oviductin ……..….…... 26 2.6.3 Hormonal Regulation of Hamster Oviductin Synthesis and Glycosylation ……………………………..……………….…. 28 vi 2.6.4 Localization of Hamster Oviductin ………………………..……... 29 2.6.5 Possible Roles of Hamster Oviductin ………………………..….... 31 2.7 Rat Oviductin ………………………………………………………..……. 33 CHAPTER 2: IN SITU AND IN VITRO IMMUNOLOCALIZATION OF OVIDUCTIN BINDING SITES ON HAMSTER UTERINE EPITHELIAL CELLS ………………………………………………………………………..……... 34 1 ABSTRACT …...…………………………………………………….………… 35 2 INTRODUCTION …...……………….…………………………….………… 37 3 HYPOTHESIS …..…………………………………………………….……… 39 4 OBJECTIVES …...…………………………………………………….……… 39 5 MATERIALS AND METHODS …..……………...……..…………...…….... 40 5.1 Animals ………………………………………………….………………... 40 5.2 Monoclonal Anti-Hamster Oviductin Antibody …….……………….…… 40 5.3 Lectin-Affinity Purification of Oviductin from Hamster Oviducts ….….... 41 5.4 Immunodetection of Purified Hamster Oviductin ………………….…..… 42 5.5 Immunohistochemical Labeling of Hamster Oviductin in the Female Reproductive Tract ………………………………………………..….…… 42 5.6 Primary Hamster Uterine Epithelial Cell Culture ……………………..….. 43 5.7 Immunoperoxidase Labeling of Cytokeratin in Cultured Primary Uterine Epithelial Cells ……………………………………………………....……. 45 5.8 Double Immunofluorescence Labeling in Cultured Primary Uterine Epithelial Cells …………………………………………………..….…….. 46 5.9 Preparation of Cultured Cells for Morphological Analysis under the Electron Microscope ………………………….………….………….……. 47 5.10 Pre-embedding Immunocytochemical Labeling of Oviductin on Cultured Primary Hamster Uterine Epithelial Cells ……………………….….……. 49 6 RESULTS …….……………………………………………….……………..... 50 6.1 Lectin-Affinity Purification of Hamster Oviductin ….……..…..……..….. 50 6.2 Immunohistochemical Labeling of Hamster Oviductin in the Female Reproductive Tract ……………………………………………..…….….... 52 6.3 Primary Cell Culture of Hamster Uterine Epithelial Cells ...…...………… 55 6.4 Detection of Cytokeratin in the Cultured Hamster Uterine Epithelial Cells …………………………………………………..….…….. 57 vii 6.5 Double Immunofluorescence Labeling of Oviductin and Cytokeratin in Cultured Primary Uterine Epithelial Cells ………………..………….…… 58 6.6 Ultrastructural Morphology of Cultured Hamster Primary Uterine Epithelial cells and Stromal Cells ……………………………...……….… 62 6.7 Pre-embedding Immunocytochemical Labeling of Oviductin on Cultured Primary Uterine Epithelial Cells ………………………………..….……... 64 7 DISCUSSION ……..…………………………………….…………………….. 68 CHAPTER 3: DETECTION OF A HAMSTER OVIDUCTIN HOMOLOGUE IN THE FEMALE RAT REPRODUCTIVE TRACT ………….………………… 75 1 ABSTRACT ………..…………………………………………………………. 76 2 INTRODUCTION ……...……..………...……………………….…………… 77 3 HYPOTHESIS …..……………………………………………….…………… 79 4 OBJECTIVES ……..……………………………………………..…………… 79 5 MATERIALS AND METHODS ……......……………………………...…….
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