RETINOIC ACID SYNTHESIS AND DEGRADATION: IMPLICATIONS FOR PROPER GERM CELL DEVELOPMENT By TRAVIS KENT A dissertation submitted in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY WASHINGTON STATE UNIVERSITY School of Molecular Biosciences DECEMBER 2015 © Copyright by TRAVIS KENT, 2015 All Rights Reserved © Copyright by TRAVIS KENT, 2015 All Rights Reserved To the Faculty of Washington State University: The members of the Committee appointed to examine the dissertation of TRAVIS KENT find it satisfactory and recommend that it be accepted. ____________________________________ Michael Griswold, Ph.D., Chair ____________________________________ Kwan Hee Kim, Ph.D. ____________________________________ Jon Oatley, Ph.D. ____________________________________ Terry Hassold, Ph.D. ii ACKNOWLEDGEMENTS I would like to take this opportunity to thank the people who have helped me get to where I am in my professional career. Much of the work presented in this document would not have been possible without the technical expertise from the Isoherranen and the Hassold-Hunt Laboratories. I would also like to thank my colleagues, both past and present in the Griswold Laboratory for their support over the years. My committee has also provided a much needed critical eye to my work. Finally, I would like to thank my friends and family who have provided me with the emotional support needed for me to persevere and thrive at Washington State. iii RETINOIC ACID SYNTHESIS AND DEGRADATION: IMPLICATIONS FOR PROPER GERM CELL DEVELOPMENT Abstract by Travis Kent, Ph.D. Washington State University December 2015 Chair: Michael D. Griswold Spermatogenesis is absolutely essential for mammalian reproduction. This highly organized and tightly controlled process is regulated, in part, by retinoic acid (RA). This essential molecule plays key roles in many aspects of spermatogenesis, namely spermatogonial differentiation, blood-testis barrier (BTB) reorganization, meiotic initiation, and spermiation. While the importance of RA is well known, the regulation of this molecule has not been well studied. RA availability is directly influenced by its synthesis and degradation, metabolized by the ALDH and CYP26 families respectively. It has recently been published that RA is present in a pulsatile manner across the spermatogenic cycle, but it is not known if these enzyme families play a role in this stage-specific availability of RA. The data presented here provides strong evidence that neither the ALDHs nor the CYP26s are regulated in a cyclic manner, suggesting that the availability of retinaldehyde, the precursor of RA, is responsible of the observed pulsatility. In addition to investigating the stage-specificity of the ALDH and CYP26 enzymes, specific inhibitors were used to investigate the effects on spermatogenesis in a high and low testicular RA environment. The data presented here shows that CYP26 inhibition causes precocious spermatogonial differentiation in the adult mouse and is able to drive synchronous spermatogenesis in the neonatal mouse. Additionally, both low and high testicular RA iv environments had abnormal effects on Sertoli cells. After inhibiting RA synthesis, BTB permeability was shown to be increased. Conversely, neonatal animals treated with exogenous RA were shown to have reduced Sertoli cell number, possibly due to premature cessation of proliferation. Finally, meiotic recombination was shown to be increased and decreased in a low and high testicular RA environment, respectively. These data highlight the importance of proper RA homeostasis within the testis, and illuminate some of the consequences of abnormal RA levels. Taken all together, the data presented in this work show the importance of understanding the expression and function of the ALDH and CYP26 enzymes in the mammalian testis. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ..................................................................................................... iii ABSTRACT ............................................................................................................................. iv LIST OF TABLES ................................................................................................................... ix LIST OF FIGURES ...................................................................................................................x DEDICATION ........................................................................................................................ xii CHAPTER 1: Checking the Pulse of Vitamin A Metabolism and Signaling during Mammalian Spermatogenesis 1. TITLE PAGE ...........................................................................................................2 2. ABSTRACT .............................................................................................................3 3. INTRODUCTION ...................................................................................................4 4. SPERMATOGENESIS ............................................................................................5 5. VITAMIN A METABOLISM .................................................................................6 6. THE ROLE OF RA DURING SPERMATOGENESIS ..........................................7 a. Spermatogonial differentiation ....................................................................7 b. Meiosis .......................................................................................................10 c. Blood-testis barrier (BTB) .........................................................................11 d. Spermiogenesis and Spermiation ...............................................................13 7. WHAT IS THE CAUSE OF STAGE-SPECIFIC RA RESPONSE? ....................15 a. Retinoid metabolism ..................................................................................15 b. Retinoid signaling ......................................................................................16 c. Retinoid transport and storage ...................................................................17 vi 8. CONCLUSIONS....................................................................................................18 9. AKNOWLEDGEMENTS......................................................................................18 10. AUTHOR CONTRIBUTION ................................................................................19 11. CONFLICTS OF INTEREST ................................................................................19 12. REFERENCES AND NOTES ...............................................................................19 13. FIGURES ...............................................................................................................28 CHAPTER 2: ALDH enzyme expression is independent of the spermatogenic cycle and their inhibition causes misregulation of murine spermatogenesis 1. TITLE PAGE .........................................................................................................32 2. ABSTRACT ...........................................................................................................33 3. INTRODUCTION .................................................................................................34 4. METHODS AND MATERIALS ...........................................................................38 5. RESULTS ..............................................................................................................48 6. DISCUSSION ........................................................................................................54 7. AKNOWLEDGEMENTS......................................................................................60 8. REFERENCES ......................................................................................................61 9. FIGURES ...............................................................................................................69 10. TABLES ................................................................................................................82 CHAPTER3: Increased testicular RA has abnormal effects on spermatogenesis 1. TITLE PAGE .......................................................................................................122 2. ABSTRACT .........................................................................................................123 3. INTRODUCTION ...............................................................................................124 vii 4. METHODS AND MATERIALS .........................................................................127 5. RESULTS ............................................................................................................133 6. DISCUSSION ......................................................................................................137 7. AKNOWLEDGEMENTS....................................................................................141 8. REFERENCES ....................................................................................................142 9. FIGURES .............................................................................................................147 10. TABLES ..............................................................................................................157 CHAPTER 4: Conclusions and Future Directions 1. CONCLUSIONS..................................................................................................159 2. FUTURE DIRECTIONS .....................................................................................164