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The Role of Gnrh-II and Its Receptor in Testicular Function Amy Desaulniers University of Nebraska - Lincoln, [email protected]

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The Role of Gnrh-II and Its Receptor in Testicular Function Amy Desaulniers University of Nebraska - Lincoln, Amy.Desaulniers@Huskers.Unl.Edu University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Theses and Dissertations in Animal Science Animal Science Department 8-2013 The Role of GnRH-II and its Receptor in Testicular Function Amy Desaulniers University of Nebraska - Lincoln, [email protected] Follow this and additional works at: http://digitalcommons.unl.edu/animalscidiss Part of the Biochemistry, Biophysics, and Structural Biology Commons, and the Other Animal Sciences Commons Desaulniers, Amy, "The Role of GnRH-II and its Receptor in Testicular Function" (2013). Theses and Dissertations in Animal Science. 150. http://digitalcommons.unl.edu/animalscidiss/150 This Article is brought to you for free and open access by the Animal Science Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Theses and Dissertations in Animal Science by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. THE ROLE OF GnRH-II AND ITS RECEPTOR IN TESTICULAR FUNCTION by Amy T. Desaulniers A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science Major: Animal Science Under the Supervision of Professor Brett R. White Lincoln, Nebraska August, 2013 THE ROLE OF GnRH-II AND ITS RECEPTOR IN TESTICULAR FUNCTION Amy Teresa Desaulniers University of Nebraska, 2013 Advisor: Brett R. White The second mammalian isoform of GnRH (GnRH-II) has been linked to regulation of cell proliferation, feed intake, and the interaction between energy balance and reproductive behavior. In contrast to the native form of GnRH (GnRH-I), GnRH-II is an inefficient modulator of gonadotropin secretion. Unlike many species, a functional receptor (GnRHR-II) specific to this ligand has been discovered in the pig that may be directly involved in testosterone production. Therefore, our objective was to identify the role of GnRH-II and its receptor in testicular function. First, there was 6-fold more GnRHR-II protein in the testis than anterior pituitary gland of boars. GnRH-II levels determined by ELISA were highest in testis (1,321 pg/ml), intermediate in pituitary (393 pg/ml) and lowest in hypothalamus (220 pg/ml) tissue homogenates. Immunohistochemistry indicated that the GnRHR-II was located on the plasma membrane of Leydig cells within the interstitium as well as germ cells. Second, immunocytochemistry demonstrated that GnRHR-IIs were localized to the neck region of spermatozoa. Consistent with this, GnRH-II was also detected in seminal plasma (225 pg/ml). Third, testicular tissue explants exposed to GnRH-II secreted testosterone similarly to hCG treated tissues, without influencing GnRHR-II or LH receptor protein levels. Finally, 4 in vivo experiments were performed on boars surgically fitted with jugular cannulae. In Exp. 1, testosterone concentrations were elevated following treatment with either a GnRH-I (D-ala6 GnRH-I) or GnRH-II (D-ala6 GnRH-II) agonist, although GnRH-II induced changes in LH levels were not correlated with testosterone production. In Exp. 2, treatment with a GnRH-I (SB-75) or GnRH-II (Trp-1) antagonist suppressed testosterone, but not LH, secretion. In Exp. 3, testosterone concentrations increased after treatment with SB-75 followed by infusion of either GnRH-I or -II, despite the inability of GnRH-II to induce LH secretion. In Exp. 4, intratesticular injection with GnRH-I, GnRH-II or saline resulted in GnRH-II-induced stimulation of testosterone production without increasing LH levels. Thus, GnRH-II may directly impact testosterone production via binding to the GnRHR-II on Leydig cells, bypassing LH production by the anterior pituitary gland. Acknowledgements First of all, I would like to thank Dr. Brett White for taking me on as a graduate student and exposing me to a wonderful balance of both basic and applied research. We spent many hours bleeding boars together, sometimes at 2 AM, which would not have been possible without his dedication to research. He also gave me great freedom to design and conduct experiments, something not many Master’s students are lucky enough to do. I am still astounded by how much I learned in these last 3 years and cannot wait to see what the next chapter brings here at UNL. I also would like to thank Drs. Andrea Cupp and Jennifer Wood for their support in completing this degree. I learned a tremendous amount in their classrooms, yes, but I also benefited from their guidance in everything from laboratory skills to presenting. They have also been extremely accommodating, always willing to meet with me despite their busy schedules. Thus, I am very grateful to have had them on my committee and look forward to working with them in the future. I also received a great deal of guidance from Drs. Clay Lents and Joe Ford. They were instrumental in performing the boar trials and gave a lot of excellent feedback on the data. Michelle McManus was also wonderful for running RIAs out at USMARC. When I first started in the lab, I knew next to nothing. So, I can safely say that Rebecca Cederberg taught me almost all my laboratory skills, and with such patience! She has also become a wonderful friend over the last 3 years, always willing to help me no matter the situation. We spent couples hours troubleshooting lab techniques together and I am so thankful to have had her guidance! I can still remember the look on her face, however, when I ripped a page out of my lab notebook to use as scrap paper….I will never do that again! If you have never met Ginger Mills, you should do so right away! She is such a great person and friend, and I am so lucky to have her on our team at UNL. The very first day I started, she took me under her wing, and I feel like I have never left! In addition to the fantastic support, I have also learned a great deal from her about my favorite livestock species and hers…pigs! Also, she was essential in our bleeding trials and always helped with a great attitude. Next, I would like to thank my parents for being such big fans these last 3 years. They have been tremendously supportive of my goals in animal reproduction research and have been there every step of the way to cheer me on. Almost every day I called my mom to update her on the day’s events. She listened to every gory detail and was so interested in my research, remembering what I told her and asking questions, even if she didn’t understand the area fully. And don’t worry mom…someday I will finish school and will move closer to you! Also, I would be remiss if I didn’t mention my wonderful boyfriend, Jake Schweitzer. He has been so great along this whole process, even through spans when we never saw each other because I was writing my thesis till midnight or bleeding pigs at 2 AM. On nights like that, he was my number one dog sitter to my boxer, Watson (aka my baby)…and for that I am SOOOO grateful. Moreover, he is always willing to listen to me blather on about testes and boar sperm…or at least I think he is listening!! A lot of this data would not have been possible without the help of several people in the lab. Bill Pohlmeier taught me how to perform IHC and immunofluorescence. Scott Kurz taught me how to do RIAs. I also had a lot of technical assistance from fellow students like John Harms, Chanho Lee and Amy Voss, Daniel Chia and Sara Stauffer. Dr. Kim Varnold and Dr. Adam Summers were also my premier statistics helpers! Finally, I have to talk about the awesome friends I made at UNL: Adam Summers, Bill Pohlmeier, Kristin Beede, Theresa Johnson, Kim Varnold, Justine Stevenson, Kevin Sargent, Michelle Semler, Renee McFee and Becky Vraspir….you guys really made Lincoln feel like home and I appreciate your friendship! vi TABLE OF CONTENTS Page LIST OF FIGURES .......................................................................................................viii LIST OF TABLES .......................................................................................................... xi CHAPTER I: Introduction Introduction ........................................................................................................... 1 CHAPTER II: Literature Review Gonadotropin-Releasing Hormone I Structure .................................................................................................... 6 Reproductive Function ............................................................................. 8 Gonadotropin-Releasing Hormone Receptor I Structure ................................................................................................... 10 Cell Signaling............................................................................................ 13 Internalization ........................................................................................... 16 Tissue Expression ..................................................................................... 17 GnRH-I and its Receptor in the Testis ...................................................... 18 Role in Cancer .......................................................................................... 22 Gonadotropin-Releasing Hormone II Structure .................................................................................................... 23 Tissue Distribution .................................................................................... 24 Reproductive Function .............................................................................. 25 Role in Behavior ......................................................................................
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