EXTRACELLULAR VESICLES, LIPIDS, AND LIPOPROTEINS IN EARLY PREGNANT SHEEP A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY University of Missouri-Columbia Division of Animal Science By ELEANORE V. O’NEIL Dr. Thomas E. Spencer, Dissertation Supervisor May 2021 The undersigned, appointed by the dean of the Graduate School, have examined the dissertation entitled EXTRACELLULAR VESICLES, LIPIDS, AND LIPOPROTEINS IN EARLY PREGNANT SHEEP presented by Eleanore O’Neil, a candidate for the degree of doctor of philosophy, and hereby certify that, in their opinion, it is worthy of acceptance. _________________________________________ Chair, Dr. Thomas Spencer _________________________________________ Dr. Kevin Fritsche _________________________________________ Dr. Rodney Geisert _________________________________________ Dr. Thomas McFadden ACKNOWLEDGEMENTS Never did I imagine I would be defending my dissertation during a pandemic! What a crazy time. This has been such a rewarding journey and I have cherished every moment here at Mizzou. I would like to acknowledge all the students, faculty, and staff for their support throughout my doctoral program. I am especially thankful to my research supervisor Dr. Thomas Spencer for giving me the opportunity to join his lab and for all the guidance and input he gave me. I would not be the scientist I am today without his mentorship. I also would like to acknowledge my committee members: Drs. Rod Geisert, Kevin Fritsche, and Thomas McFadden for their support and the wonderful discussions we had during my time here. I want to give a special shoutout to Dr. Geisert for his unwavering support of me. I must extend the deepest gratitude for the members of Team Uterus, past and present: Drs. Kelsey Brooks, Joao Moraes, Andrew Kelleher, Pramod Dhakal, Kosta Simintiras, as well as Jacob Pearson, Wang Peng, Megan McClean, Clay Mitchell, and Jessica Milano-Foster. I consider Drs. M Sofia Ortega and Harriet Fitzgerald from the Spencer Lab to be both mentors and friends who really helped shape my academic journey. Most notably, Dr. Greg Burns and David Todd were my unending allies in the annual craziness that is Sheep Season. It has been a pleasure to call them my colleagues and friends. Finally, sincere appreciation and love for my fellow grad students, especially those in the Geisert Lab (Ashley Meyer, Caroline Pfeiffer, and Destiny Johns) and the Ortega Lab (Jason Rizo, Kelsey Clark, and Katy Stoecklein). They spent so many of their evenings at the barn, so ii many mornings at sheep surgeries, and plenty of afternoons laughing at lunch with me. It really takes a village. Finally, to my crazy, loving family (‘The Oneks’)! My parents, Susan and Dennis, are my real-life heroes and my biggest cheerleaders, and my three older siblings, Iver, Lisabeth, and Stefan, and my future sister-in-law Shataakshi, are my best friends. My family never doubted me during all these years and even flew out to the middle of Missouri (leaving Florida, Washington, North Carolina, and New York) to spend Christmas with me and Will so I could stay with the sheep! It was worth it because two of our family Christmas photos featured us posing with sheep. And of course, thank you to my partner William Deng, who has been with me (and will be with me) through it all. I could have not asked for a better partner, and personally, I think he deserves a Ph.D. in sheep reproduction too! iii TABLE OF CONTENTS ACKNOWLEDGEMENTS…………………………………………………………...…ii LIST OF FIGURES…………………………………………..……………….…..……vii LIST OF TABLES…………………………………………..…………………………...x LIST OF ABBREVIATIONS…………………………………………………………...xi ABSTRACT……………………………………………………………………………xiii CHAPTER I: LITERATURE REVIEW……………………………………………………………...1 The Uterus………………………………………………………………………1 Uterine histoarchitecture………………………………….…………….1 Estrous cycle…………………………………………………………….2 Early Pregnancy………………………………………………………………..4 Conceptus development, implantation, & placentation……………..4 Maternal recognition signaling ………………………………………..7 Regulation of the Uterine Environment……………………………………8 Progesterone regulation of the endometrium…………………...……8 IFNT regulation of the endometrium………………………………..…9 Histotroph and uterine luminal fluid………………………………….10 Extracellular Vesicles……………………………………………………..…12 Biosynthesis of extracellular vesicles………………………………..12 Extracellular vesicle cargo & uptake…………………………………15 Extracellular vesicles in the uterine lumen……………………….…17 Metabolites and Lipids ……………………………………………………..21 Metabolites in the uterine lumen…………………………………..…22 Lipids in the uterine lumen……………………………………………24 Conceptus use of metabolites and lipids ……………..………….…27 iv Prostaglandins ……………………………………………………………….30 Biosynthesis of prostaglandins ………………………………………30 Prostaglandins in the uterine lumen…………………………………34 Lipoproteins…………………………………………………………………..38 Biosynthesis of lipoproteins……………………………………..……39 Lipoproteins and apolipoproteins in the uterine lumen……….……44 II: CHARACTERIZATION AND REGULATION OF EXTRACELLULAR VESICLES IN THE LUMEN OF THE OVINE UTERUS……………………..……..47 Abstract………………………………………………………………………..48 Introduction……………………………………………………………………49 Materials and methods……………………………………………………...51 Results…………………………………………………………………………61 Discussion…………………………………………………………………….71 Acknowledgements………………………………………………………….81 Supplemental information………………………………………………….82 III: EXTRACELLULAR VESICLE EFFECTS ON THE ENDOMETRIUM OF SHEEP…………………………………………………….……….……....…………..83 Abstract………………………………………………………………………..84 Introduction……………………………………………………………………85 Materials and methods………………………………………………………86 Results…………………………………………….………...…………………92 Discussion……………………………………………..………...……………98 Acknowledgements…………………………………………...……………103 Supplemental information…………………………………...……………103 IV: INSIGHTS INTO THE LIPIDOME AND PRIMARY METABOLOME OF THE UTERUS FROM DAY 14 CYCLIC AND PREGNANT SHEEP……....…………104 Abstract………………………………………………………………………105 Introduction……………………………………………………………….…106 Materials and methods………………………………………………….…108 v Results…………………………………………….……………………….…112 Discussion……………………………………………..………………….…123 Acknowledgements…………………………………………...……………131 Supplemental information………………………………………………...131 V: PROSTAGLANDIN-ENDOPEROXIDE SYNTHASE 2 IS NOT REQUIRED FOR PREIMPLANTATION OVINE CONCEPTUS DEVELOPMENT IN SHEEP……………………………………………………………………..………….132 Abstract………………………………………………………………………133 Introduction…………………………………………………………….……134 Materials and methods……………………………………………….……136 Results…………………………………………………………………..……144 Discussion………………………………………………………………...…150 Acknowledgements…………………………………………………...……154 Supplemental information…………………………………………...……154 VI: BIOLOGICAL ROLE OF APOA1 IN EARLY PREGNANCY IN SHEEP.…156 Abstract………………………………………………………………………157 Introduction…………………………………………………………….……158 Materials and methods……………………………………………….……160 Results…………………………………………………………………..……167 Discussion………………………………………………………………...…173 Acknowledgements…………………………………………………...……177 Supplemental information……………………………………………...…177 SUMMARY AND CONCLUSIONS…………………………………………………178 REFERENCES……………………………………..……...…………………………189 VITA……………………………………………………………………………………223 vi LIST OF FIGURES I: LITERATURE REVIEW Figure 1. Histoarchitecture of the uterus……………………..…………...….………2 Figure 2. Early conceptus development……………………………………...………6 Figure 3. Regulation of the endometrial epithelium during early pregnancy……10 Figure 4. Synthesis, secretion, and uptake of extracellular vesicles………….…14 Figure 5. Working hypothesis on the role of extracellular vesicles (EV) in early pregnancy..……………………………………..………………………………………19 Figure 6. Schematic of prostaglandin (PG) biosynthesis…………...…….………32 Figure 7. Lipoprotein classes………...………………………………………………39 Figure 8. Lipoprotein interactions……………………………………………………43 II: CHARACTERIZATION AND REGULATION OF EXTRACELLULAR VESICLES IN THE LUMEN OF THE OVIEN UTERUS Figure 1. Extracellular vesicle (EV) number in the uterine lumen of day 14 cyclic and pregnant sheep…………………..…………………………………..……………61 Figure 2. Proteomic analysis of extracellular vesicles (EV) isolated from the uterine lumen of day 14 cyclic and pregnant sheep…………………………………….……65 Figure 3. Phospholipids in extracellular vesicles (EV) isolated from the uterine lumen of day 14 cyclic and pregnant sheep…………………….…………...………67 Figure 4. Ceramides, acylcarnitines, and free fatty acids (FFAs) in extracellular vesicles (EV) isolated from the uterine lumen of day 14 cyclic and pregnant sheep……………………………………………………………………………………68 Figure 5. Effect of extracellular vesicles (EV) isolated from the uterine lumen of day 14 cyclic and pregnant sheep on ovine trophectoderm (oTr) cell proliferation………………………………………………………..……………………70 Figure 6. Effect of extracellular vesicles (EV) isolated from the uterine lumen of day 14 cyclic and pregnant sheep on IFNT production by ovine trophectoderm (oTr) cells…………………………………………………………….…………………71 vii III: EXTRACELLULAR VESICLE EFFECTS ON THE ENDOMETRIUM OF SHEEP Figure 1. Summary of endometrial transcriptome analyses…………….……..…93 Figure 2. Enrichment analysis of differentially expressed transcripts in CEV infused ewes……..…….…..…….…..…….…..…….…..……………………...…….94 Figure 3. Prostaglandins were elevated in the uterine lumen of CEV infused ewes compared to PEV infused ewes…..………….……………………………………….96 Figure 4. Infusion of PEV prevented upregulation of uterine ESR1…….....…….97 IV: INSIGHTS INTO THE LIPIDOME AND PRIMARY METABOLOME OF THE UTERUS FROM DAY 14 CYCLIC AND PREGNANT SHEEP Figure 1. Triglycerides (TAG) in the endometrium from day 14 cyclic and pregnant sheep…………………………….……………………………………………...…….114