University of Rhode Island DigitalCommons@URI Open Access Master's Theses 2013 Are Full-Length mRNA In Bos taurus Spermatozoa Transferred to the Oocyte During Fertilization? Elizabeth Jane Anderson University of Rhode Island, [email protected] Follow this and additional works at: https://digitalcommons.uri.edu/theses Recommended Citation Anderson, Elizabeth Jane, "Are Full-Length mRNA In Bos taurus Spermatozoa Transferred to the Oocyte During Fertilization?" (2013). Open Access Master's Theses. Paper 84. https://digitalcommons.uri.edu/theses/84 This Thesis is brought to you for free and open access by DigitalCommons@URI. It has been accepted for inclusion in Open Access Master's Theses by an authorized administrator of DigitalCommons@URI. For more information, please contact [email protected]. ARE FULL-LENGTH mRNA IN Bos taurus SPERMATOZOA TRANSFERRED TO THE OOCYTE DURING FERTILIZATION? BY ELIZABETH J ANDERSON A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN BIOLOGICAL AND ENVIRONMENTAL SCIENCE UNIVERSITY OF RHODE ISLAND 2013 MASTER OF SCIENCE IN BIOLOGICAL AND ENVIRONMENTAL SCIENCE THESIS OF ELIZABETH J ANDERSON APPROVED: Thesis Committee: Major Professor Becky L. Sartini, Ph.D. Katherine Petersson, Ph.D. Steven Irvine, Ph.D. Nasser H. Zawia, Ph.D. DEAN OF THE GRADUATE SCHOOL UNIVERSITY OF RHODE ISLAND 2013 ABSTRACT This thesis focuses on the discovery of full-length mRNA transcripts in Bos taurus spermatozoa. The primary aim of this study is to identify and validate full- length mRNA primarily from RNA-Sequencing of bovine spermatozoa. The secondary aim is to determine if full-length spermatozoal transcripts are delivered to the oocyte at fertlilization, allowing for future studies to track their inheritance from paternal sources to the embryo. The main hypothesis of this thesis is that full-length mRNA transcripts exist within the spermatozoal transcript profile in Bos taurus. The secondary hypothesis is that if spermatozoal mRNA is functional after fertilization, then full-length transcripts should be present in the early stage embryo. To examine these hypotheses, this thesis is divided into three main chapters. The first is a literature review, discussing the process of spermatogenesis, the unique properties of spermatozoal mRNAs, including some hypothesized functions of spermatozoal mRNAs. A summary of a new technique, RNA-Sequencing, will be discussed in this review as well as comparisons to previous literature techniques for identifying mRNA transcripts of interest. The second chapter is the manuscript published in the journal Biology of Reproduction in January 2013, co-first-authored by Christopher Card. This manuscript uses the technique RNA-Seq to examine the transcript profile of nine Bos taurus bulls, and highlights several transcripts of interest for further study. This study found 6,166 total transcripts, and performed Gene Ontology analysis of the transcripts to categorize them into functional categories for further examination, the top most category of interest being translation. The third chapter of this thesis is a manuscript in preparation, formatted for submission to the journal of Molecular Reproduction and Development. This manuscript evaluates twenty four target mRNA transcripts to see if they are full- length. These transcripts were identified through four main methods: their location on the Y chromosome, their high expression in the RNA-Seq data set from chapter 2, their presence in Gene Ontology categories of interest from chapter 2, and their discovery from previous literature studies. Sixteen transcripts are found to be full- length, eight are degraded, and four have alternative polyadenylation ends. In conclusion, several full-length transcripts were found in this study, which have the potential to create functional proteins downstream in the fertilized oocyte. Several transcripts were also proved to be degraded in the mature spermatozoa. This has confirmed the need for this type of study, and elucidates new transcript targets for further research to pursue. ACKNOWLEDGMENTS I would like to thank Dr. Becky Sartini for her continuous support both scientifically and emotionally, throughout the process of developing my master’s thesis. Additionally, the members of my lab have made my years here at URI much more enjoyable and productive: Justin Richard, Erin Aparicio, Krystle Schultz, and Jazmin Zamberlan. Particular to thanks to Christopher Card, who has been an excellent collaborator and mentor throughout my masters degree. Without the personal support of my amazing husband, and love of my life, Edward Anderson, my commitment to these manuscripts would have been much more difficult. My parents and sister have been a continual source of support throughout this process as well. Last, but certainly not least, I would like to thank all of the mentors that I have had the pleasure of working with during my time at URI. To my committee members, Dr. Katherine Petersson, Dr. Steven Irvine, and Dr. Ingrid Lofgren, I appreciate the time, effort, and patience involved with reading and working through my thesis. I would also like to thank the teachers I’ve worked with: Fred Launer, Emma Kaiser, and Marta Gomez-Chiarri. iv PREFACE The content of this thesis is subdivided into two different manuscripts. The first is a manuscript co-authored with Christopher Card and published in January 2013 in the journal “Biology of Reproduction.” Liz was a co-first author on this manuscript with a primary focus on identification of full-length transcripts in the bovine spermatozoal transcript profile. She was also responsible for all gene ontology analysis. Liz was responsible for tables 3, 5, 6, and figure 3, and collaborated with Chris Card on figures 1, 3, 4, and tables 1, 2, and 4. The writing and editing of the manuscript was shared equally between Chris and Liz. The second manuscript here is in preparation for submission to the journal “Molecular Reproduction and Development.” This work is done entirely by Liz. v TABLE OF CONTENTS ABSTRACT .................................................................................................................. ii ACKNOWLEDGMENTS .......................................................................................... iv PREFACE ..................................................................................................................... v LIST OF FIGURES ................................................................................................... xii CHAPTER 1: LITERATURE REVIEW ................................................................... 1 I. Introduction ............................................................................................................ 1 II. Gametogenesis ....................................................................................................... 1 II. A. Spermatogenesis ........................................................................................... 2 III. What is spermatozoal mRNA? .............................................................................. 5 III. A. Composition ................................................................................................ 5 III. B. Potential Functions of Spermatozoal mRNA .............................................. 7 III.B.1. Spermatozoal survival in the female reproductive tract and fertilization ............................................................................................................................ 8 III.B. 2. Early embryogenesis ............................................................................ 8 III. B.2.a. Oocyte activation ........................................................................... 9 III. B.2.b. Protamines: chromatin repackaging & regulation ....................... 10 III. B. 2. c. Oocyte meiotic division ............................................................. 11 III. B. 2. d. Embryo imprinting .................................................................... 12 III. B. 2. e. Epigenetic influences ................................................................. 12 III. B. 2. f. Regulating proper embryo development .................................... 13 vi III. B. 3. Spermatozoal mRNA use as a fertility assay .................................... 14 IV. Full-Length mRNA Transcripts .......................................................................... 15 V. RNA-Seq: a global transcript discovery method ................................................. 17 VI. Hypotheses.......................................................................................................... 19 VII. Aims ................................................................................................................... 19 VIII. References ........................................................................................................ 19 CHAPTER 2: BIOLOGY OF REPRODUCTION MANUSCRIPT ..................... 28 Abstract .................................................................................................................... 30 Introduction .............................................................................................................. 31 Materials and Methods ............................................................................................ 33 Spermatozoa Samples .......................................................................................... 33 RNA Isolation .....................................................................................................