ROLE OF LEPTIN IN REGULATING THE BOVINE HYPOTHALAMIC-GONADOTROPIC AXIS A Dissertation by MARCEL AMSTALDEN Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY December 2003 Major Subject: Physiology of Reproduction ROLE OF LEPTIN IN REGULATING THE BOVINE HYPOTHALAMIC-GONADOTROPIC AXIS A Dissertation by MARCEL AMSTALDEN Submitted to Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved as to style and content by: Gary L. Williams Thomas E. Spencer (Co-Chair of Committee) (Co-Chair of Committee) Paul G. Harms Thomas H. Welsh, Jr. (Member) (Member) Rajesh C. G. Miranda John W. McNeill (Member) (Head of Department) December 2003 Major Subject: Physiology of Reproduction iii ABSTRACT Role of Leptin in Regulating the Bovine Hypothalamic-Gonadotropic Axis. (December 2003) Marcel Amstalden, D.V.M., Universidade Estadual Paulista, Botucatu, Brazil; M.S., Texas A&M University Co-Chairs of Advisory Committee: Dr. Gary L. Williams Dr. Thomas E. Spencer The physiological mechanisms through which nutrition mediates its effects in controlling reproduction are not well characterized. Both neural and endocrine components have been implicated in the communication of nutritional status to the central nervous system. Leptin, a hormone synthesized and secreted mainly by adipocytes, is heavily involved in this communication network. The objectives of studies reported herein were 1) to determine the effects of short-term restriction of nutrients on circulating leptin, leptin gene expression in adipose tissue, and leptin receptor (LR) gene expression in the adenohypophysis of ovariectomized cows; and 2) to investigate the responsiveness of the hypothalamic-adenohypophyseal (AP) axis of fasted and non-fasted cattle to leptin. Studies demonstrated that circulating concentrations of leptin and leptin gene expression in subcutaneous adipose tissue are decreased by fasting. Although 2 to 3 days of fasting did not affect patterns of release of luteinizing hormone (LH), cerebroventricular infusions of leptin increased mean iv circulating concentrations of LH in fasted, but not normal-fed cows, without affecting frequency or amplitude of pulses of LH. In vitro studies were conducted to determine whether the in vivo effects of leptin could be accounted for at the hypothalamic and/or AP levels. Leptin did not affect the release of gonadotropin-releasing hormone (GnRH) from hypothalamic-infundibular explants from either normal-fed or fasted cattle. Moreover, leptin did not affect the basal release of LH from bovine AP cells or AP explants from normal-fed cows. However, leptin induced a higher basal release of LH from AP explants of fasted cows and increased GnRH-stimulated release of LH from AP explants of normal-fed cows. Results demonstrate that leptin acts directly at the AP level to modulate the secretion of LH, and its effects are dependent upon nutritional status. Cellular mechanisms associated with the increased responsiveness of gonadotropes to leptin in fasted cows were investigated. Expression of LR and suppressor of cytokine signaling-3 (SOCS-3) in the adenohypophysis did not account for the increased responsiveness of fasted cows to leptin. Therefore, although leptin clearly stimulates the hypothalamic-gonadotropic axis in nutrient-restricted cattle, it is unclear why cattle maintained under neutral or positive energy balance are resistant to leptin. v DEDICATION This manuscript is dedicated to the memory of my brother, Marcos, who has always been with me and to my daughter, Sophia, who has fulfilled my life. vi ACKNOWLEDGMENTS I would like to express my sincere gratitude and appreciation to Dr. Gary L. Williams, who has for the last six years provided guidance, advice, and support for my academic and scientific endeavors at Texas A&M University. I am very fortunate to be part of Dr. Williams’ program, which provided an exceptional opportunity for training and career planning. I would like to extend my appreciation to Dr. Paul G. Harms, for the extraordinary support given to my academic and personal life. Appreciation is also extended to Dr. Thomas E. Spencer for providing support and expertise on our research projects, to Dr. Thomas H. Welsh, Jr., for the time and effort invested in helping with our research, and to Dr. Rajesh C. G. Miranda for serving as committee member. Thanks to Dr. Nancy H. Ing for providing a great opportunity for training, and Dr. Robert C. Burghardt and Dr. Luc Berghman for supporting our projects. Thanks also to Dr. Randy L. Stanko, Dr. Steven E. Nizielski, and Dr. Suresh Pillai for their assistance in our studies. I would like to acknowledge Dr. Duane Keisler for providing the ovine leptin and leptin-receptor cDNA and performing leptin RIA, Dr. Jerry Reeves for LH antisera, Dr. James Dias for FSH antisera, Dr. Terry Nett for GnRH antisera, and Dr. A.F. Parlow (National Pituitary Hormone Program) for pituitary hormones. Appreciation is also extended to those people that provided their time and friendship to assist in my research: Michelle Garcia, Dr. Dorota Zieba, Joanna Gallino, Stephanie Morton, Marlon Maciel, Melvin Davis, Randle Franke, Mark Besancon, vii Marsha Green, Brad Thedin, Trey Lopez, Justin Kinnamon, Pat Chen, Tina Bryan, Dr. JoAnn Fleming, Youngsok Choi, Dr. Jianbo Hu, Dr. Kanako Hayashi, Sekoni Noel, Allison Gray, Karen Carpenter, Seokwoom Kim, Nahum Puebla, James Totten, Cindy Balog, Yuhua Zhang, Dana Dean, Kerry Dean, Sherry Kelly, and Kitty Tate. With all my heart, a special gratitude to my wife, Kátia, who has been wonderful and supportive throughout these years. A special thanks also to my parents, Francisco and Beatriz, Tia Nena, and the remainder of my family for their support and encouragement. viii TABLE OF CONTENTS Page ABSTRACT ………………………………………………………………..… iii DEDICATION ………………………………………………………….......... v ACKNOWLEDGMENTS …………………………………………………… vi TABLE OF CONTENTS ………………………………………….................. viii LIST OF FIGURES ………………………………………………………...... xii LIST OF TABLES …………………………………………………………… xv CHAPTER I INTRODUCTION …………………………………………………… 1 II LITERATURE REVIEW ……………………………………………. 4 The hypothalamic-gonadotropic axis ………………………………… 4 Hypothalamic-adenohypophyseal complex ………………...... 4 Regulation of hypothalamic secretion of GnRH …………....... 7 Regulation of adenohypophyseal release of gonadotropins ..… 12 Nutritional effects on hypothalamic-gonadotropic function …………. 18 Metabolic and neuroendocrine mediators of nutritional effects on the hypothalamic-gonadotropic axis ……………………….………… 20 Glucose as a metabolic fuel ………………………………….. 20 Insulin as a metabolic hormone ……………………………… 21 The GH, IGF-I, and IGF-binding protein axis ……………….. 22 Neuropeptide Y as neurotransmitter regulating GnRH neurons ……………………………………………………….. 23 Melanocyte-stimulating hormone …………………………..... 24 Agouti-related peptide ………………………………………... 25 Ghrelin ………………………………………………………... 26 Galanin ……………………………………………………….. 27 Orexin ………………………………………………………… 28 Leptin as a putative signal of nutritional status to the hypothalamic-gonadotropic axis ……………………………………... 29 ix TABLE OF CONTENTS (cont.) CHAPTER Page III CENTRAL INFUSION OF RECOMBINANT OVINE LEPTIN NORMALIZES PLASMA INSULIN AND STIMULATES A NOVEL HYPERSECRETION OF LUTEINIZING HORMONE AFTER SHORT-TERM FASTING IN MATURE BEEF COWS .…………… 36 Introduction …………………………………………………………... 36 Material and methods ………………………………………………… 38 Animal model and intracerebroventricular delivery of recombinant oleptin ……………………………………….. 38 Procedures ……………………………………………………. 39 Radioimmunoassay and colorimetric assays …………………. 43 Northern blot analysis ………………………………………… 44 Statistical analysis ……………………………………………. 44 Results ………………………………………………………………... 45 Effects of short-term fasting on leptin gene expression in adipose tissue; on circulating insulin, leptin, and glucose; and on pulsatile LH release ………………………………..…. 45 Effects of ICV infusions of oleptin on plasma insulin, leptin, glucose, and LH secretion ……………………………………. 48 Discussion ……………………………………………………………. 53 IV EFFECTS OF LEPTIN ON GONADOTROPIN-RELEASING HORMONE RELEASE FROM HYPOTHALAMIC-INFUNDIBULAR EXPLANTS AND GONADOTROPIN RELEASE FROM ADENOHYPOPHYSEAL PRIMARY CELL CULTURES: FURTHER EVIDENCE THAT FULLY-NOURISHED CATTLE ARE RESISTANT TO LEPTIN …………………………………………… 59 Introduction …………………………………………………………... 59 Materials and methods ……………………………………………….. 60 Experiment 1 …………………………………………………. 60 Experiment 2 …………………………………………………. 63 Experiment 3 …………………………………………………. 63 Radioimmunoassays …………………………………………. 64 Statistical analysis ……………………………………………. 65 Results ………………………………………………………………... 66 Experiment 1 …………………………………………………. 66 Experiment 2 …………………………………………………. 66 x TABLE OF CONTENTS (cont.) CHAPTER Page Experiment 3 …………………………………………………. 66 Discussion ……………………………………………………………. 71 V LEPTIN ACTS AT THE BOVINE ADENOHYPOPHYSIS TO ENHANCE BASAL AND GONADOTROPIN-RELEASING HORMONE-MEDIATED RELEASE OF LUTEINIZING HORMONE: DIFFERENTIAL EFFECTS ARE DEPENDENT UPON NUTRITIONAL HISTORY …………………………………. 75 Introduction …………………………………………………………... 75 Materials and methods ……………………………………………….. 77 Animal model and procedures ………………………………... 77 Effects of leptin on basal and GnRH-mediated release of LH from AP explants ………………………………………… 78 Effects of leptin on GnRH secretion from the isolated medial basal hypothalamus-infundibular complex …………………... 80 Radioimmunoassays ………………………………………….. 81 Statistical analysis ……………………………………………. 82 Results ………………………………………………………………..