EVIDENCE FOR THE ROLE OF YWHA IN MOUSE OOCYTE MATURATION A thesis submitted To Kent State University in partial Fulfillment of the requirements for the Degree of Master of Science By Ariana Claire Detwiler August, 2015 © Copyright All rights reserved Except for previously published materials Thesis written by Ariana Claire Detwiler B.S., Pennsylvania State University, 2012 M.S., Kent State University, 2015 Approved by ___________________________________________________________ Douglas W. Kline, Professor, Ph.D., Department of Biological Sciences, Masters Advisor ___________________________________________________________ Laura G. Leff, Professor, PhD., Chair, Department of Biological Sciences ___________________________________________________________ James L. Blank, Professor, Dean, College of Arts and Sciences i TABLE OF CONTENTS List of Figures ……………………………………………………………………………………v List of Tables ……………………………………………………………………………………vii Acknowledgements …………………………………………………………………………….viii Abstract ……………………………………………………………………………………….....1 Chapter I Introduction…………………………………………………………………………………..2 1.1 Introduction …………………………………………………………………………..2 1.2 Ovarian Function ……………………………………………………………………..2 1.3 Oogenesis and Folliculogenesis ………………………………………………………3 1.4 Oocyte Maturation ……………………………………………………………………5 1.5 Maternal to Embryonic Messenger RNA Transition …………………………………8 1.6 Meiotic Spindle Formation …………………………………………………………...9 1.7 YWHA Isoforms and Oocyte Maturation …………………………………………...10 Aim…………………………………………………………………………………………..15 Chapter II Methods……………………………………………………………………………………...16 Collection of oocytes and eggs…………………………………………………………..16 Collection of morulae and blastocysts …………………………………………………..17 Messenger RNA isolation and cDNA synthesis for YWHA gene expression…………..18 ii Polymerase chain reaction and gel electrophoresis for YWHA gene expression………..19 Gene sequencing ………………………………………………………………………...20 Quantitative polymerase chain reaction …………………………………………………20 Protein isolation for pull-down ………………………………………………………….21 SDS-PAGE and Western blot for protein pull-down ……………………………………22 Chapter III Results ………………………………………………………………………………………24 1. Do the levels of YWHA gene expression change during oocyte maturation? …….....24 Background and rationale ……………………………………………………………….24 1.1 Detecting YWHA isoform gene expression by RT-PCR ……………………….25 1.2 Sequencing YWHA isoform PCR products ……………………………………..26 1.3 Measuring levels of gene expression through oocyte maturation by quantitative PCR………………………………………………………………………………34 2. Is YWHAH a binding partner for CDC25B in ovary, oocytes, and eggs? …………..45 Background and rationale ……………………………………………………………….45 2.1 Detecting the binding of CDC25B to YWHAH in oocytes and eggs through pull-down experiments …………………………………………………………..46 Summary ………………………………………………………………………………………..48 iii Chapter IV Discussion …………………………………………………………………………………..49 References ……………………………………………………………………………………...55 Appendix of Abbreviations ……………………………………………………………………61 iv LIST OF FIGURES CHAPTER I Figure 1. Follicular development ………………………………………………………………...4 Figure 2. Mouse oocyte maturation ……………………………………………………………...5 Figure 3. Simplified diagram of proteins involved in oocyte maturation from arrest at prophase (left) to release from meiotic arrest (right)……………………………………...7 Figure 4. Immunoprecipitation of CDC25B phosphatase and co-immunoprecipitation of YWHA proteins with CDC25B ……………………………………………………………..12 Figure 5. Microinjection of antisense morpholino oligonucleotides ………………………...…14 CHAPTER III Figure 6. Expression of YWHA isoform mRNA in oocytes and eggs of two mouse strains …..26 Figure 7. H2AfZ (NM_016750.3) mRNA Sequence …………………………………………...27 Figure 8. YWHAH (NM_011738.2) mRNA Sequence ………………………………………...28 Figure 9. YWHAB (NM_018753.6) mRNA Sequence ……………………………………..….29 Figure 10. YWHAE (NM_009536.4) mRNA Sequence ……………………………………….30 Figure 11. YWHAG (NM_018871.3) mRNA Sequence ……………………………………….31 v Figure 12. YWHAQ (NM_011739.3) mRNA Sequence ……………………………………….32 Figure 13. YWHAZ (NM_011740.3) mRNA Sequence ……………………………………….33 Figure 14. Fold changes between oocyte and egg for the YWHAH isoform …………………..35 Figure 15. Fold changes between oocyte and egg for the YWHAE isoform …………………..36 Figure 16. Fold changes between oocyte and egg for the YWHAG isoform …………………..37 Figure 17. Fold changes between oocyte and egg for the YWHAQ isoform ...………………...38 Figure 18. Fold changes between oocyte and egg for the YWHAZ isoform …………………...39 Figure 19. Fold changes between morula and blastocyst for the YWHAH isoform ...…….…...43 Figure 20. Pull down of YWHAH and CDC25B proteins in oocyte and egg ………………….47 vi LIST OF TABLES CHAPTER II Table 1. Primers used to detect presence of each YWHA isoform in RT-PCR ………………19 Table 2. Primers used to detect each YWHA isoform in qPCR ………………………………21 CHAPTER III Table 3. Quantitative PCR average CT values for each YWHA isoform and Luciferase, ΔCT values, and ΔΔCT values ………………………………………………………………40 Table 4. Quantitative PCR Average CT values for YWHAH isoform and Luciferase, ΔCT values, and ΔΔCT values calculated from morula and blastocyst ……………………..44 vii ACKNOWLEDGEMENTS I would like to express my sincere gratitude to all who have helped me to accomplish this goal, especially to my advisor, Dr. Kline. Without his patient guidance and encouragement, I would not have been able to complete this thesis. I have learned so much from his mentoring and I will be forever grateful for the time that we spent working together. I would also like to extend my gratitude towards my lab mates, past and present, Santanu De and Alaa Eisa. I am so glad that we became such close friends and peers in the short time we had here at Kent State together. I am also very grateful to Dr. Vijayaraghavan and his lab members, Suranjana Goswami, Rahul Bhattacharya, Shawn Davis, Nidaa Awaja, Sabyasachi Sen, Tejasvi Dudiki, and Cameron Brothag, as we worked closely as sister labs on our projects. I am also grateful and appreciative to the undergraduate students who helped me with a few projects, Patricia Marthey, Michele Fabian, and Isabella Cruz. I must also express my great thanks to Dr. Jenny Marcinkievicz for serving on my committee and fostering my love of teaching. Many thanks to all of the other professors, graduate students, and staff here at Kent State who have assisted me, guided me, and encouraged me in my pursuit of this degree. And last but not least, thank you to my parents, my brother, the rest of my extended family, and all of my close friends who have supported me and encouraged me during this process. viii ABSTRACT Processes essential to the growth and differentiation of oocytes, such as meiosis and spindle formation, are dependent upon intricate interactions between key proteins. The Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation (YWHA) family of proteins have been found to be essential to vertebrate cell cycle regulation and development. There are seven isoforms, each encoded by a different gene. Previous studies have shown that these proteins are involved in maintenance of prophase I arrest in the GV-intact oocyte, as well as proper spindle formation. Real-time and quantitative PCR methods were employed to study the relative levels of expression of these proteins’ transcripts in the mouse oocyte and egg. Protein pull-down experiments were also used to investigate the interaction of the key phosphatase in meiosis resumption, CDC25B, and the YWHAH protein in oocytes and eggs. PCR results confirm the presence of at least six of the seven isoforms in both oocytes and eggs of two strains of mice, and suggest a decrease in relative expression of the transcripts through oocyte maturation. The pull- down results confirm the binding and interaction of YWHAH with CDC25B. This study increases the knowledge available on the YWHA proteins, and provides a strong basis for further studies. 1 CHAPTER I INTRODUCTION 1.1 Introduction Processes essential to the growth and differentiation of germ cells, such as meiosis and spindle formation, are dependent upon intricate interactions between key proteins. The Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation protein (YWHA) family of proteins has been found to be essential to vertebrate cell cycle regulation and development. Recent studies have shown that these proteins also play vital roles in oocyte maturation, meiotic spindle formation, and early embryonic development. These processes are necessary and required to produce fertilizable eggs, and determining the function and expression of the YWHA proteins in immature oocytes, mature eggs, and the developing embryo is essential to understanding these pathways. 1.2 Ovarian Function The mammalian ovary is a complex organ, characterized by continuous growth and differentiation throughout the animal’s maturation. The ovary is the primary reproductive organ for the female, and carries out two main functional roles. First, the ovary is the site of differentiation of the mature oocyte and after maturation, releases the fertilizable egg (McGee 2 and Hsueh, 2000). Second, the ovary synthesizes and secretes hormones essential for follicle development, maintenance of the reproductive tract, and regulating the menstrual/estrous cycle (Hirshfield, 1991). In mice, the ovary is a small organ, filled with germ cells and stroma at birth. During the first week after birth, the ovary grows and differentiates exponentially, and by 7 days old, oocyte growth and follicle development has begun (Peters, 1969). Ovulation and thus, sexual