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Epigenetic Reprogramming, Apoptosis, and Developmental Competence in Cloned Embryos Utah State University DigitalCommons@USU All Graduate Theses and Dissertations Graduate Studies 8-2019 Epigenetic Reprogramming, Apoptosis, and Developmental Competence in Cloned Embryos Laura A. Moley Utah State University Follow this and additional works at: https://digitalcommons.usu.edu/etd Part of the Animal Sciences Commons Recommended Citation Moley, Laura A., "Epigenetic Reprogramming, Apoptosis, and Developmental Competence in Cloned Embryos" (2019). All Graduate Theses and Dissertations. 7571. https://digitalcommons.usu.edu/etd/7571 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. EPIGENETIC REPROGRAMMING, APOPTOSIS, AND DEVELOPMENTAL COMPETENCE IN CLONED EMBRYOS by Laura A. Moley A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Animal, Dairy, and Veterinary Sciences Approved: ______________________ ____________________ S. Clay Isom, Ph.D. Abby D. Benninghoff, Ph.D. Major Professor Committee Member ______________________ ____________________ John Stevens, Ph.D. Trista Strauch, Ph.D. Committee Member Committee Member ______________________ ____________________ Aaron Thomas, Ph.D. Richard S. Inouye, Ph.D. Committee Member Vice Provost for Graduate Studies UTAH STATE UNIVERSITY Logan, Utah 2019 ii Copyright © Laura Moley 2019 All Rights Reserved iii ABSTRACT Epigenetic Reprogramming, Apoptosis, and Developmental Competence in Cloned Embryos by Laura A. Moley, Doctor of Philosophy Utah State University, 2019 Major Professor: Dr. S. Clay Isom Department: Animal, Dairy, and Veterinary Sciences Cloning through somatic cell nuclear transfer (SCNT) remains highly inefficient twenty years after the first demonstration of the technology. The technology could be more routinely utilized to enhance animal agriculture production by increasing efficiency and decreasing cost through the selection of embryos that are most likely to succeed following transfer. We proposed the use of a non-toxic, non-invasive caspase activity reporter, SR-FLICA, to quantitatively assess embryo competency prior to transfer. We hypothesized that following SCNT, a majority of embryos undergo altered epigenetic reprogramming compared to in vivo embryos, resulting in aberrant gene expression and incidentally increased levels of apoptosis. Conversely, a fraction of the embryos undergo proper genome reprogramming, resulting in correct gene expression and low levels of apoptosis. Using porcine embryos, the non-toxic nature of SR-FLICA was validated on oocytes and embryos. No correlation between relative level of apoptosis and cell number was found in blastocysts. DNA methylation and gene expression patterns were analyzed through reduced-representation bisulfite sequencing and RNA-seq to provide global iv patterns in high and low apoptosis SCNT blastocysts. No differences in global DNA methylation patterns were found between the high apoptosis SCNT, low apoptosis SCNT, and in vivo produced embryos. Comparing high apoptosis and low apoptosis porcine SCNT blastocyst identified 4,346 100 bp tiles that were differentially methylated and 413 genes that were differentially expressed. Hierarchical clustering of differentially methylated tiles separated the high apoptosis from low apoptosis SCNT samples. However, hierarchical clustering of differentially expressed genes did not separate samples based on apoptosis. Gene ontology analysis identified no significantly enriched terms or KEGG pathways from genes with a differentially methylated tile near the transcription start site or from differentially expressed genes. Embryo competency was evaluated using day 7 bovine SCNT blastocysts that were transferred into females for evaluation of pregnancy establishment. While SR-FLICA did not hinder pregnancy rates, apoptosis score was not an indicative measure for pregnancy establishment. Together, these data support a stochastic nature of epigenetic reprogramming following SCNT and reinforce the necessity to identify embryos most likely to be successful due to proper epigenetic reprogramming in order to increase SCNT efficiency. (265 pages) v PUBLIC ABSTRACT Epigenetic Reprogramming, Apoptosis, and Developmental Competence in Cloned Embryos Laura A. Moley Cloning through somatic cell nuclear transfer (SCNT) remains highly inefficient twenty years after the first demonstration of the technology with the birth of Dolly. By increasing efficiency by selecting the embryos early in development that are most likely to succeed following transfer into a surrogate mother, the technology could be more routinely utilized to enhance animal agriculture production. SCNT is believed to be highly inefficient as a result of incorrect DNA methylation and gene expression that are accumulated because of the SCNT technique. We proposed the use of a non-toxic, non- invasive detector of cell death, to quantitatively assess embryo competency prior to embryo transfer. We believed we could use SR-FLICA to identify the embryos with low levels of cell death as a result of proper DNA methylation and gene expression. By analyzing the whole embryo, differences in gene expression and DNA methylation were identified in embryos with high and low levels of cell death. However, the level of cell death did not prove to be a reliable indicator of embryo quality in predicting pregnancy outcome. This data supports the commonly held hypothesis that DNA methylation and gene expression after SCNT have random defects as a result of the random nature of resetting the DNA for embryo development. More research is required to identify the embryos which will prove to be successful following SCNT and embryo transfer. vi ACKNOWLEDGMENTS This project could not have happened without financial support from the United States Department of Agriculture and the Agriculture and Food Research Initiative Competitive Grant no. 2013-67015-20964 from the USDA National Institute of Food and Agriculture. Additional funding from the Animal, Dairy, and Veterinary Science department and the department of Research and Graduate Studies at Utah State University was critical for the success of the project. I am indebted to Dr. Clay Isom for the patience, guidance, and training provided over my time at Utah State University. No one could ask for a better mentor through their graduate career. He has shown me, through example, what a good scientist should be. Additionally, advice, support, and direction from my graduate student committee greatly aided my research and writing. Thank you to Dr. Abby Benninghoff, Dr. Aaron Thomas, Dr. John Stevens, and Dr. Trista Strauch for your time and talents throughout my graduate career. This project would not have been possible without the help of numerous others. I am beyond grateful to all those who kept this project going along the way. The support I received from the Isom lab made all the difference, including help from Harry Zhao, Sameer, Alhojaily, Farnaz Malekjahani, Sierra Heywood, Connor Waldron, and Samantha Krieger. Dr. Rakesh Kaundal and Rousselene Larson provided bioinformatic analysis that was far beyond my capacity to conquer. Dr. Qinggang Meng and Dr. Ying Liu expertly performed micromanipulations to create cloned embryos. The White lab group, specifically Misha Regouski and Jake Keim, performed bovine cloning and organized embryo transfers after many failed attempts of porcine embryo transfers. The veterinary group of Utah State University, including Dr. Rust Stott, Dr. Lexi Sweat, and Sarah Behunin, performed the vii embryo transfers. Finally, a huge thank you to the farm crew who took care of the animals, day in and day out. Lastly, I could have never achieved all of this without the love and support of my family and friends. Words cannot express the gratitude I feel towards my parents, who prioritized and were the driving force behind my education from day one. This achievement is just as much yours as it is mine. I am so incredibly lucky to have met my fiancée along this journey. He was always there to celebrate the experiments that worked, but much more often hear the frustrations of those that did not. Steve- I truly could not have done this without you. Finally, to the many friends- Sydney, Amanda, Cara, Sam, Nikole, Laura, and many more- who been there every step of the way, I am eternally thankful. Laura A. Moley viii CONTENTS Page ABSTRACT ...................................................................................................................... iii PUBLIC ABSTRACT .........................................................................................................v ACKNOWLEDGMENTS ................................................................................................ vi LIST OF TABLES ..............................................................................................................x LIST OF FIGURES ........................................................................................................ xiii CHAPTER I. REVIEW OF LITERATURE .................................................................................1 Assisted Reproductive Technologies .............................................................. 1 Somatic Cell Nuclear Transfer ....................................................................... 6 Epigenetics ...................................................................................................
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