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The Role of Dmrt1 in Postnatal Testis Differentiation

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The Role of Dmrt1 in Postnatal Testis Differentiation THE ROLE OF DMRT1 IN POSTNATAL TESTIS DIFFERENTIATION By Copyright 2012 Valentine A. Agbor B.Sc., University of Buea, 2002; M.S., New Mexico Highlands University, 2006 Submitted to the graduate degree program in Molecular and Integrative Physiology and to the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Committee: Leslie L. Heckert, Ph.D., Chair Joseph S. Tash, Ph.D. Kenneth R. Peterson, Ph.D. T. Rajendra Kumar, Ph.D V. Gustavo Blanco, M.D., Ph.D. Dissertation defended: August 31, 2012 The Dissertation Committee for Valentine A. Agbor certifies that this is the approved version of the following dissertation: THE ROLE OF DMRT1 IN POSTNATAL TESTIS DIFFERENTIATION Dissertation Committee: Leslie L. Heckert, Ph.D., Chair Date Approved: September 4, 2012 ii Abstract DMRT1 (doublesex and maleabnormal-3 related transcription factor-1) is a transcription regulator that is expressed in Sertoli cells and germ cells of the testis, where it directs mammalian postnatal testis differentiation. It is currently the only mammalian gene within the sex determination and sex differentiation genetic pathways that is evolutionary conserved and shares a cysteine-rich DNA binding motif (DM-domain) with doublesex in the fruit fly and maleabnormal-3 in the roundworm. The work presented in this dissertation revealed numerous novel roles of DMRT1, including Sertoli cell-specific rescue, structural function in the testis, cell-specific pathways and targets, and a probable regulation by NANOS2 in germ cells; thus providing important insight into how DMRT1 regulates testis development. Recombinogenic engineering was invaluable in the generation of a novel Sertoli cell-rescue (Dmrt1-/-;tg) mouse model, which is a DMRT1-deficient germ cell model that was produced by breeding Dmrt1-null (Dmrt1-/-) mice with Wt1-Dmrt1 transgenic mice. The Wt1-Dmrt1 transgenic mice express a rat Dmrt1 cDNA in gonadal supporting cells by directing it from the Wilms' tumor locus carried on a yeast artificial chromosome transgene. Characterization of the Sertoli cell rescue mice revealed Sertoli cell-specific function of DMRT1 in the maintenance of seminiferous tubule morphology, autonomous and non-autonomous dose-dependent gene regulation, Sertoli cell differentiation, maintenance of testis size and sperm progressive motility, organization of testis-specific adherens junctions and maturation of Leydig cells and thus regulation of testosterone production. Furthermore, cell-specific direct and indirect pathways and targets regulated by DMRT1 in Sertoli cells and germ cells were identified using global gene expression profiles combined with ChIP-Seq. Analyses of the gene profiles identified potential functions for DMRT1 in vesicular transport, ubiquitination and RNA binding. Similarly, analysis of ChIP-Seq data identified 150 iii targets with the majority of DMRT1 binding sites located in introns, indicating that DMRT1 preferentially binds to distal regulatory regions. Finally, direct protein-protein interaction was shown between DMRT1 and NANOS2, an evolutionary conserved RNA-binding protein that plays a key role in male germ cell differentiation, by inhibiting entry into meiosis. NANOS2 was shown to act upstream, interact, partially co-localize and repress the in vitro transcriptional activity of DMRT1 in germ cells. Overall, these experiments uncovered several questions and new cell-specific roles of DMRT1 in testis differentiation. These findings and their implications are discussed further in this dissertation within the general scope of reproductive biology and the more defined scope of postnatal testis differentiation. iv Acknowledgement This dissertation symbolizes my negligible contribution that nourishes an infinite dedication to science, which is congenital and supported by my mentor, Dr. Leslie Lynn Heckert, whose leadership was invaluable to this chef-d'oeuvre. Her thoughtfulness and tolerance to let me learn through mistakes; her patience to teach me to think, plan, write and perform as a scientist is highly appreciated and I am truly indebted. Gratitude goes to my parents, siblings, cousins, nieces, nephews, girlfriend and in-laws whose encouragement kept the candle burning during good and bad times. Student life would have been boring without the countless lifetime friends I have made during graduate school, especially Elizabeth Dille and Jitu George, this life-changing experience has come and gone, but our shared relationships have made us resilient and will last forever. For the numerous student groups and governing bodies that I was involved with, especially the Physiology Society provided valuable distractions and opportunities to improve my leadership skills. This opportunity was conceivable because of the incessant support for the graduate student community by Dr. Paul Cheney, Chair of the Department of Molecular and Integrative Physiology. Moreover, for the administrative staff members of the University who spent uncountable hours working together with these groups to improve the student experience at the University of Kansas Medical Center. I am grateful to the members of Dr. Heckert’s laboratory, past and present, with and from whom I have learned so much including: Dr. Brian Hermann, Dr. Ning Lei, Kaori Hornbaker, Dr. Tatiana Karpova, Dr. Ravichandran Kumarasamy, Dr. Mathew Anway, Dr. Shixin Tao, v Anindita Chatterjee, Daren Rice, Dr. Barbara Sotolongo, Lovella Tejada, Jitu George and Elizabeth Dille. My sincere gratitude to those whose technical assistance greatly influenced the success of the experiments reported in this dissertation including Dr. David Albertini, Dr. William Kinsey, Barbara Fegley, Clark Bloomer, Dr. Sumedha Gunewardena, Dr. Stan Svojanovsky, Dr. In-Hee Lee and Dr. Mahesh Visvanathan. I am appreciative for the continuous assistance from staff members of the Department of Molecular and Integrative Physiology and Center for Reproductive Sciences, past and present including: Jennifer Wallace, Linda Carr, Shari Standiferd, Liz Meng, Linda Spears and Barbara Shull without whom this work would not have come to fruition. Finally, I am indebted to my student advisory committee, whose impartial feedbacks, recommendations and guidance were invaluable in accomplishing my research aims, experimental design, and professional development; all of which were vital elements of my graduate education. vi Table of contents Abstract .......................................................................................................................................... iii Acknowledgement .......................................................................................................................... v Table of contents ........................................................................................................................... vii List of Figures and Tables............................................................................................................... x List of Abbreviations ................................................................................................................... xiv Chapter 1: Literature Review .......................................................................................................... 1 Introduction ............................................................................................................................. 2 Reproduction ........................................................................................................................... 2 Types of sex determination systems ........................................................................................ 2 Sexual differentiation in Mammals ......................................................................................... 6 Gonad development ................................................................................................................. 7 Germ cell development .......................................................................................................... 10 The postnatal testis ................................................................................................................ 13 DMRT1 and DM FAMILY ................................................................................................... 15 Conclusion ............................................................................................................................. 20 Chapter 2: A Wt1-Dmrt1 transgene restores DMRT1 to Sertoli cells of Dmrt1-/- testes; a novel model of DMRT1-deficient germ cells (Submitted to Biology of Reproduction)........................ 27 Abstract .................................................................................................................................. 28 Introduction ........................................................................................................................... 29 vii Materials and Method ............................................................................................................ 32 Results ................................................................................................................................... 38 Discussion .............................................................................................................................. 47 Chapter 3: DMRT1 in Sertoli cells regulates the organization and formation of ectoplasmic specialization................................................................................................................................
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