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Characterization and Evaluation of Androgen-Binding Protein, Sex Hormone-Binding Globulin, and Thyroxine- Binding Globulin in the Horse

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Characterization and Evaluation of Androgen-Binding Protein, Sex Hormone-Binding Globulin, and Thyroxine- Binding Globulin in the Horse University of Kentucky UKnowledge Theses and Dissertations--Veterinary Science Veterinary Science 2018 CHARACTERIZATION AND EVALUATION OF ANDROGEN-BINDING PROTEIN, SEX HORMONE-BINDING GLOBULIN, AND THYROXINE- BINDING GLOBULIN IN THE HORSE Blaire O'Neil Fleming University of Kentucky, [email protected] Digital Object Identifier: https://doi.org/10.13023/etd.2018.353 Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Fleming, Blaire O'Neil, "CHARACTERIZATION AND EVALUATION OF ANDROGEN-BINDING PROTEIN, SEX HORMONE-BINDING GLOBULIN, AND THYROXINE-BINDING GLOBULIN IN THE HORSE" (2018). Theses and Dissertations--Veterinary Science. 37. https://uknowledge.uky.edu/gluck_etds/37 This Master's Thesis is brought to you for free and open access by the Veterinary Science at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Veterinary Science by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Blaire O'Neil Fleming, Student Dr. Alejandro Esteller-Vico, Major Professor Dr. Daniel K. Howe, Director of Graduate Studies CHARACTERIZATION AND EVALUATION OF ANDROGEN-BINDING PROTEIN, SEX HORMONE-BINDING GLOBULIN, AND THYROXINE-BINDING GLOBULIN IN THE HORSE _________________________________________ THESIS __________________________________________ A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in the College of Agriculture, Food and Environment at the University of Kentucky By: Blaire O’Neil Fleming Lexington, Kentucky Director: Alejandro Esteller-Vico, DVM, Ph.D Lexington, Kentucky Copyright © Blaire O’Neil Fleming 2018 ABSTRACT OF THESIS CHARACTERIZATION AND EVALUATION OF ANDROGEN-BINDING PROTEIN, SEX HORMONE-BINDING GLOBULIN, AND THYROXINE-BINDING GLOBULIN IN THE HORSE The objectives of this study are to characterize two carrier proteins in the horse that significantly decrease in humans following anabolic androgenic steroid administration: sex hormone-binding globulin (SHBG) and thyroxine-binding globulin (TBG). For SHBG characterization, qPCR, RNA sequencing, and immunohistochemistry were performed on testes and equine livers. Free and total testosterone immunoassays were utilized to confirm the presence of a carrier protein in equine circulation. SHBG was detected in the testes using qPCR, RNA sequencing, and IHC, indicating the presence of the isoform androgen-binding protein (ABP). SHBG was not detected in any liver samples. Evidence of a carrier protein was shown by free testosterone being significantly lower than the total testosterone that was detected in stallions (p<0.0001) and pregnant mares (p<0.0001). TBG characterization was completed using an equine specific TBG ELISA. Equine serum was analyzed across seasons, reproductive statuses, sexes, and ages. TBG concentrations were also measured following anabolic steroid administration (Stanozolol) and increased endogenous androgen production via hCG administration in stallions and aromatase inhibition via Letrozole administration in pregnant mares. TBG did not significantly differ across season, reproductive status, sex, or age Alterations of androgen concentrations did not result in any significant changes to circulating TBG concentrations. KEYWORDS: Anabolic, Biomarker, Sex Hormone-Binding Globulin, Thyroxine- Binding Globulin, Horse Blaire O’Neil Fleming 8-11-2018 CHARACTERIZATION AND EVALUATION OF ANDROGEN-BINDING PROTEIN, SEX HORMONE-BINDING GLOBULIN, AND THYROXINE-BINDING GLOBULIN IN THE HORSE By Blaire O’Neil Fleming Dr. Alejandro Esteller-Vico Director of Thesis Dr. Daniel K. Howe Director of Graduate Studies 8-11-2018 I would like to dedicate this thesis to my family and my fiancé, for always believing in the scientist inside me. Mom, Dad, and Sissy; from the incessant questions of a child to the investigations of scholar, you have encouraged my passion and curiosity and helped to guide me to the path I am on today. DJ, you took a leap of faith by helping me follow my dreams. Without you, the completion of this thesis would not have been possible. ACKNOWLEDGEMENTS The following thesis, while an individual work, would not have been possible without the insight and support from so many people. First, I must thank Dr. Alejandro Esteller-Vico (Alex), my Thesis Chair and primary advisor. We worked together past many bumps in the road to complete a body of research that I am quite proud of. His assistance with this entire process has been invaluable. Alex has taught me so much in the realm of academia, as well as in life in general. He and this entire experience have made a lasting impact that have changed me as a scientist, for the better. Next, I must thank Dr. Barry Ball for challenging me to be the most informed scientist that I can be. His guidance has caused me to become much more of an investigative researcher by looking into every possible area surrounding the topic at hand. Being a part of his equine reproduction laboratory at the Maxwell H. Gluck Equine Research Center has been an absolute privilege and I am honored to have had the opportunity to work with him. I must also thank Dr. Tom Curry for being a part of my thesis committee. Dr. Curry jumped on board at an interesting point during the completion of my degree and his insight from the human side of reproduction was very much appreciated. Of course, none of this would have been possible without the financial support provided by the Albert G. Clay Endowment, Shapiro Endowment, and Kentucky Equine Drug Research Council. I thank these funding sources for the opportunity to be able to pursue this degree. In addition to my committee and funding sources, the completion of this thesis would not have been possible without my friends and family. I have to thank my reproduction laboratory for assisting me in every step of this thesis. From teaching me iii new laboratory techniques to laughs during lunch, these amazing people made this process an absolute joy. Dr. Kirsten Scoggin, your patience is everlasting and you never once hesitated to answer the absurd amount of questions that I was able to come up with over the past 3 years. Our entire lab owes our functionality and technique knowledge to you. Dr. Yatta Boakari, you became my other half. We both always seemed to being going through the same struggles at the same time and we made it through together. Your friendship will continue to be precious to me. Dr. Shavahn Loux, you were beyond helpful not only in interpreting RNA sequencing data, but also in being a sounding board for any concerns I may have had. Michelle Wynn, you were a wonderful source of knowledge and friendship inside and outside of the lab. Dr. Mariano Carossino, you went out of your way to guide me through immunohistochemistry, no matter the day of the week. Dr. Hossam El-Sheikh Ali, you are probably the kindest person I have ever met. You were always ready with words of encouragement and a smile. Dr. Pouya Dini, you would never allow me to present an idea without asking the hard questions. This made me a better presenter and researcher and for that, I thank you. Dr. Carleigh Fedorka, you became my big sister in science over the years. You were a wonderful friend and teacher, whether it be on the farm, in the lab, or in life. I started this journey with new colleagues and am leaving my Master’s degree with family. I am so thankful to have had the opportunity to meet every single one of you. Now, it is time to thank the family that the good Lord gave me. To my parents, Margaret and George Fleming, thank you for always pushing me to strive for the best. You encouraged me to follow a scientific path from a young age and it grew into a passion that has taken me on some unexpected roads. I would not be where I am today if iv you both had not instilled the importance of knowledge in me. To my papa, Tim O’Neil, thank you for always telling me to dream the impossible dream and fight the unbeatable foe. I am proud to say that I conquered my Master’s with your words of wisdom always present in my heart and mind. To my sister, Lauren (aka Sissy), having you in Lexington, KY with me during my graduate career was an honest blessing.
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