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Ann Arbor, Ml 48106 PARTIAL CHARACTERIZATION AND PURIFICATION OF STEROIDOGENIC FACTORS IN THYMIC EPITHELIAL CELL CULTURE- CONDITIONED MEDIUM Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Mehmet Uzumcii, D.V.M., M.S. The Ohio State University 1994 Dissertation Committee: Young C. Lin, DVM, Ph.D. Approved by Robert L. Hamlin, DVM, Ph.D. Joseph S. Ottobre, Ph.D. Advisor Yasuko Rikihisa, Ph.D. Dept. Vet. Physio/Pharm. To Meryem ACKNOWLEDGEMENTS In the name o f Allah, the Most Merciful, the Most Beneficent. Limitless praise belongs to Allah by whose mercy this study has been completed. Then, I express my sincere appreciation to Dr. Young C. Lin, for his support, guidance and insight throughout this study. My appreciation also goes to the other members of my advisory committee Drs. Joseph S. Ottobre, Yasuko Rikihisa and Robert L. Hamlin for their invaluable help. I also thank Dr. David Brigstock for the help that he provided during my study. All kinds of assistance that were provided to me by the staff, especially Ms. Margie Maxwell and Mrs. Arlene Myers, and my colleagues, Drs. Shigeo Akira, Yan Gu, Hiroshi Ohmura, George Chang, Serdar Coskun, Yun Fu Hu, Sam Kulp, Falah Shidaifat, William Chang, Halit Canatan, Moustafa El-Banna, and Muhammad Mushtaq is gratefully appreciated. I appreciate the financial support that was provided for the early part of this study by The Scientific and Technical Research Council of Turkey (TUBlTAK). I would also like to express my gratitude to my parents for everything that they provided for me. Finally and above all, I offer sincere thanks to my wife, Selma, for her continuous support, understanding and encouragement during my study. I thank my children, VITA March 31, 1964....... Bom-Karapinar, Konya, Turkey. 1986 ......................... Doctor of Veterinary Medicine (D.V.M.), University of Ankara, College of Veterinary Medicine, Turkey. 1986-198 8 .............. Doctoral Student, Department of Reproduction and Artificial Insemination, University of Ankara, College of Veterinary Medicine. 1987- 1988 ............. Veterinarian, Ministry of Agriculture, Ankara, Turkey. 199 0......................... M.S. in Veterinary Physiology and Pharmacology, The Ohio State University. 1991 -present Graduate Research Associate, The Ohio State University. PUBLICATIONS: Uziimcii M and Lin YC (1990) Factor(s) from rat thymic cell culture conditioned medium (TCM) stimulate(s) progesterone secretion (P) in cultured rat granulosa cells. Biol Reprod 42: Suppl. # 1, pl63. Uztimcii M. and Lin YC, (1991) Evidence for thymic factors (TF) in the regulation of steroidogenesis in cultured rat granulosa cells (RGC). Program and Abstract of the 73rd Annual Meeting of Endocrine Society, p368. Moh PP, Uziimcii M. Chang GCJ, Hu YF, Brewer ML, and Lin YC, (1991) Effects of estrogen on covalent binding of gossypol to microsomal proteins in rats. Biol Reprod 45: Suppl # 1, pl31. Uziimcii M. Akira S, and Lin YC, (1992) Stimulatory effect of thymic factor(s) on steroidogenesis in cultured rat granulosa cells. Life Sciences 51:1217-1228. Uziimcii M. and Lin YC, (1992) Gossypol (GP) inhibits thymic factors (TF)-stimulated steroidogenesis in cultured rat granulosa cells (GC). Biol Reprod 46: Suppl. #1, p i68. Ohmura H, Chang WY, Uziimcii M. Coskun S, Akira S, Araki T, and Lin YC, (1993) Transforming growth factor B1 stimulates progesterone production in cultured granulosa cells from gonadotropin-primed adult rats. In: Leung PCK, Hsueh AJW, Friesen HG (eds), Molecular Basis of Reproductive Endocrinology, Springer-Verlag, New York, 1993; pp 215- 221. Uziimcii M. Ohmura H, Chang CJG, Araki T, and Lin YC, (1993) Factor(s) in thymic cell culture conditioned medium (TCM) stimulate(s) steroidogenesis in cultured porcine granulosa cells. FASEB J 7: A20. Fehim EM, Lin YC, Golder JM, Bruggemeier RW, and Uziimcii M. (1993) Effect of an aromatase inhibitor on steroid hormone secretion in cultured rat granulosa cells (RGC). FASEB J 7: A617. Ghosh PK, Uziimcii M. York JP, and Lin YC, (1993) Effects of galanin on lactic acid and estradiol production by Sertoli cells isolated from immature rats. Program and Abstracts of the 75th Annual Meeting of Endocrine Society, pl02. Akira S, Ohmura S, Uziimcii M. Araki T, Lin YC, (1994) Gossypol inhibits aromatase activity in cultured porcine granulosa cells. Theriogenology 41:1489-1497. Uziimcii M. and Lin YC, (1994) Characterization of stimulatory action of thymic factor(s) on basal and gonadotropin-induced steroidogenesis in cultured rat granulosa cells. Mol Cel Endocrinol 105:209-216. Kulp S, Uziimcii M. Mushtaq M, Rikihisa Y, and Lin YC, (1994) Inhibition of rat Sertoli cell function in vitro by a larval tapeworm excretory/secretory product. FASEB J 8: A576. Uziimcii M. Brigstock DR and Lin YC (1994) Rat thymic epithelial cell culture conditioned medium (TCM) contains two distinct steroidogenic factors. Biol Reprod 50: Suppl # 1, p74. Coskun S, Uziimcii M. Lin YC, Friedman Cl and Alak B (1994) Porcine cumulus cell steroidogenesis is regulated by oocytes. Biol Reprod 50: Suppl # 1, pl44. v FIELD OF STUDY Major Field: Veterinary Physiology and Pharmacology Studies in Reproductive Endocrinology and Immunology. TABLE OF CONTENTS DEDICATION.............................................................................................................. ii ACKNOWLEDGEMENTS...................................................................................... iii VITA........................................................................................................................... iv TABLE CONTENTS................................................................................................ vii LIST OF FIGURES................................................................................................... ix LIST OF PLATES........................................................................................................ xii LIST OF TABLES................................................................................................... xiii INTRODUCTION....................................................................................... 1 CHAPTER I. THYMIC EPITHELIAL CELL CULTURE CONDITIONED-MEDIA (TCM) STIMULATES STEROIDOGENESIS IN CULTURED RAT GRANULOSA CELLS....................................................... 21 Introduction ........................................................................ 21 Materials and methods ....................................................... 23 Results................................................................................. 30 Discussion .......................................................................... 33 Summary............................................................................ 37 Figures ................................................................................ 39 Plates................................................................................... 64 vii CHAPTER H. CHARACTERIZATION OF THE STIMULATORY ACTIONS OF THYMIC FACTORS ON BASAL AND GONADOTROPIN-INDUCED STEROIDOGENESIS IN CULTURED RAT GRANULOSA CELLS.......................... 77 Introduction ..................................................................... 77 Materials and methods ................................................... 79 Results............................................................................. 85 Discussion ....................................................................... 88 Summary......................................................................... 93 Figures ............................................................................. 95 CHAPTER HI TWO DISTINCT STEROID-MODULATING FACTORS WERE IDENTIFIED IN RAT THYMIC EPITHELIAL CELLS CULTURE-CONDITIONED MEDIA