The Role of Exogenous Somatotropin, Ovariectomy and Extracellular Matrix in Bovine Mammary Gland Development
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The Role of Exogenous Somatotropin, Ovariectomy and Extracellular Matrix in Bovine Mammary Gland Development Brandy Patrice Huderson Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Animal Science, Dairy Committee: Dr. R. Micheal Akers (Chair) Dr. Mark D. Hanigan Dr. Eric A. Wong Dr. William R. Huckle Dr. Ronald E. Pearson January 27, 2010 Blacksburg, Virginia Keywords: heifer, mammary, growth hormone, ovariectomy, MAC-T The Role of Exogenous Somatotropin, Ovariectomy and Extracellular Matrix in Bovine Mammary Gland Development Brandy Patrice Huderson ABSTRACT The highly regulated maturation of the mammary gland is poorly understood. Our studies were designed to further characterize the role of ovarian hormones, growth hormone (GH)/IGF axis proteins and extracellular matrix (ECM) in the growth and development of prepubertal mammary glands. Prepubertal heifers were injected with either exogenous GH or subjected to ovariectomy (OVX). Mammary parenchyma (PAR) and mammary fat pad (MFP) were harvested for DNA, protein, lipid, and western blot analysis. Remaining tissues were preserved for histological staining or snap frozen for quantitative real-time PCR. We examined 13 genes that work in conjunction with the extracellular matrix to regulate mammary proliferation and morphogenesis. Administration of GH, while impacting composition of MFP, had no effect on expression of the selected genes; there was a decrease in expression of fibronectin in PAR. Ovariectomy had no effect on gene expression in MFP but decreased expression of epimorphin, a potent regulator of morphogenesis, in PAR. In both experiments, the presence of a 55 kDa band corresponding to androgen converting enzyme aromatase was detected but its expression was unaffected. In another study, we used in vitro cell culture to evaluate the role of ECM in mammary gland maturation and employed quantitative real-time PCR to evaluate gene expression profiles of select genes involved in proliferation and differentiation. Expression of Rac1 was decreased in response to bovine insulin (BI) but increased on collagen I (Col). Expression of aldehyde dehydrogenase was decreased in BI and serum on plastic and on Col in the presence of BI. Expression of IGF binding proteins (BP) 3, -4, and -6 were decreased in the presence of serum on laminin (LM). Also, IGF-BP2 expression was decreased on Col while IGF-BP6 was increased on LM with BI. Clusterin, a ubiquitous non-adhesive ECM protein was not affected by ECM substrate but did increase over time. In conclusion, we propose that the mammary gland is not able to respond to GH at this age and that while OVX did effect the expression of some genes, the presence of aromatase maintained local estrogen concentrations. Furthermore, ECM alone is insufficient to regulate mammary gland development and growth. ACKNOWLEDGEMENTS I would like to take this opportunity to express my deepest gratitude to Dr. Akers, my major advisor and committee chair. Thank you for your seemingly endless patience. Thank you for taking a chance on me and giving me this opportunity. The past four and a half years have been the best of my life and I owe that to you. Your support and generosity has allowed me to grow as a researcher and as an academic. It has been an honor to work in your lab. I would like to also thank my dissertation committee. Dr. Pearson you willingness to sit with me and repeatedly explain statistics and SAS as whole is greatly appreciated. Dr. Huckle, your warm smile was greatly appreciated when I was imagining my academic career flying out of the window. Dr. Hannigan, thank you for the time you have taken out to help me. Your vast breathe of knowledge is both impressive and a bit intimidating, but I appreciate the kindness that you have shown me. Dr. Wong, I owe you special thanks not only for serving on my dissertation committee or for the help that you have provided me but also for the many opportunities that I have been afforded with PREP. To my dear lab sisters, lab just wasn’t the same without you here. I am so sad that our paths have forked but I am so proud of both of you. Ladies, your patience and willingness to conquer SAS for me can never be repaid but I am certainly alright with spending the rest of life trying. No other lab will ever come close to what we had here. It was privilege to work next to such phenomenonal women. I will never forget our times here in lab, making messes and trying to figure out the simplest of task. I am excited about our next adventures and look forward to seeing both of you at least once a year, every year. I am so humbled and honored to have been a part of the PREP/IMSD family. You were the first family I had after coming to VT and I am so thankful for the soft landing. To Dr. Smith, thank you for taking a chance on me. You have been a guiding light in my time here at VT and I thank you. Many of the opportunities that I have had here at VT have been a direct result of the PREP/IMSD family and I only hope that I have represented you well. Soror Kershaw, thank you for all your help! You have been there for me through thick and thin. I wish you and your family all the best. Thank you, Pattie Boyle, for all your help and support. To this day, whenever I hear “Celebration” or “Ladies Night” I just want get up and dance. Continue to be happy and beautiful. Cathy, your problem solving super strength probably saved my dissertation more than once. Thanks for all your help and time especially with taking pictures. I have no doubt that you will soar through your master’s program whenever the time is right. While I am grateful to all of DASC faculty I am especially grateful to Dr. G and Isis. I am not sure what I did to deserve it but your unrelenting belief and confidence in me was the bright spot in many of my days. Thank you for every piece of advice you ever gave me, every kind word and every smile. Cindy, please continue to be wonderful. To Julie and Becky, thank you for all your help. Had it not been for you ladies I am not sure that I would have been enrolled each semester I never knew there was so much fun to be had in a Dairy Science department. To the grad students, I say thank you for accepting an “outsider” and making me feel like this was where I belonged. Kto, Agustin, Jobi and Joe, best of luck in all that you do. Megan your very interesting and often times hilarious questions were classic and will forever be etched on my mind. Ivory, you probably don’t know it, but your friendship during one of the darkest times in my life saved iii me. I am so happy for you and wish you many years of wedded bliss. I can’t wait to see the tall, chubby babies! All of this would have been impossible if it were not for the love and support of my family. Ma, thank you for daily calls and encouragement even when you didn’t know what you were encouraging me to do. A big thank you for taking temporary custody of Salaam and Nyah, I know they can be a handful but they love their grandmother very much. Big Pimp, thank you for being the best daddy a girl could have, you never judged me, just laughed at me, you never chastised me, just laughed at me. You remind me that nothing is that important. Bigurl, Diva, you are one-half of the best things our parents ever did. Of all the people in my life I admire you the most. You are a beautiful woman and I am exceedingly proud to know you much less be your sister. You challenge me and steal my things but I wouldn’t have it any other way. Diva, I can’t wait to toast you as Dr. Huderson, you will take this world by storm! To Boogers and Gal, it has been an honor to have such strong black women in my life. You both have seen many sunsets yet your strength and wisdom have never been eclipsed. Thank you, both, for making me feel like the best granddaughter in the world. I can’t promise a husband and kids, but I can promise to always strive to make you proud of me. To my Nubian Princesses, Coco and Meme, you are the inspiration in my life. All that I do is for you, to show that there is a better way. You can accomplish anything that you put your minds to. And when you fall short I will always be there to lift you up. Thank you for your patience, I love you both. Ley and Nitra, wow, I have officially known you ladies most of life and I can’t picture, nor do I want to, my future without you. You have seen me at my worst and have stood by me the entire time. We have seen the tragedy of death and the miracle of birth together. We have grown into beautiful women together and are a force to be reckoned with, as long as no one sees the antics that happen behind closed doors. A toast to us, the Holee Trinity. It is amazing, most of us spend the majority of our time here in the NRV begging to leave, but when that time comes we find that it is the hardest thing to do.