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Microrna Expression and Function During Porcine Oocyte Maturation and Early Embryonic Development Elane Courtney Wright Iowa State University

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Microrna Expression and Function During Porcine Oocyte Maturation and Early Embryonic Development Elane Courtney Wright Iowa State University Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2012 MicroRNA expression and function during porcine oocyte maturation and early embryonic development Elane Courtney Wright Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Animal Sciences Commons, and the Molecular Biology Commons Recommended Citation Wright, Elane Courtney, "MicroRNA expression and function during porcine oocyte maturation and early embryonic development" (2012). Graduate Theses and Dissertations. 12522. https://lib.dr.iastate.edu/etd/12522 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. MicroRNA expression and function during porcine oocyte maturation and early embryonic development by Elane Courtney Wright A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Co-majors: Animal Science and Molecular, Cellular and Developmental Biology Program of Study Committee: Jason W. Ross, Major Professor Lloyd L. Anderson Jeffrey J. Essner Aileen F. Keating Christopher K. Tuggle Iowa State University Ames, Iowa 2012 Copyright© Elane Courtney Wright, 2012. All rights reserved. ii TABLE OF CONTENTS LIST OF TABLES…………………………………………………………………………..vii LIST OF FIGURES…………………………………………………………………………viii NOMENCLATURE…………...……………………………………………………………..xi ACKNOWLEDGEMENTS……..………………………………………………………….xiii ABSTRACT……………………...…………………………………………………………xv Chapter 1. Introduction……………………………………………………………………….1 Chapter 2. Review of Literature…………………………………………………………….3 Utilization of pigs in agriculture…………………………………………….………...3 Utilization of pigs for biomedical research……………………………….………….3 Estrous cycle……………………………………………………………….………….5 Estrus synchronization …………………………………………………………….....9 Follicle growth ad recruitment…………………………………………….………...10 The Oocyte………………………………………………………………………..….11 Metaphase II arrest…………………………………….……………………………..13 Germinal vesicle breakdown………………………………….……………………..14 In vitro vs. In vivo maturation ………………………………………………………..15 Fertilization…………………………………………………………….…………….16 Early embryo development and uterine migration in the pig…………………...…...18 Embryo elongation and implantation…………………………………….…..………19 iii Small RNAs in the oocyte, early embryo and elongating conceptus…….……….…22 RNA…………………………………………………………………….….………...22 MicroRNA……………………………………………………………….……..……22 MiRNA in the pig……………………………………………………………………24 MiRNA in the oocyte and early embryo………….……………………….…….…..26 MiRNA in the elongating pig conceptus……………………………….…….……...27 MiRNA-21…………………………………………………………………………..28 Summary…………………………………………………………………………….32 Chapter 3. MicroRNA21 Expression and Regulation of PDCD4 during In vitro Maturation of Porcine Oocytes.............................................................................................33 Abstract………………………………………………………………………………34 Introduction…………………………………………………………………………..35 Materials and Methods……………………………………………………………….37 In vitro maturation…………………………………………………………...37 MIR21 expression in oocytes with and without LH and FSH during in vitro maturation…………………...…………………………………………38 MIR21 expression in oocytes cultured with and without cumulus cells…….38 MIR21 inhibition during In vitro maturation ……………………………….39 Quantitative RT-PCR of pooled porcine oocytes……………………………40 PDCD4 Western blot analysis……………………………………………….41 Immunostaining of GV and MII oocytes…………………………………….42 In situ hybridization of MIR21 in the developing follicle…………….……..43 Statistical Analysis…………………………………………………………...43 iv Results………………………………………………………………………………..44 MIR21 expression is temporally regulated during porcine cumulus oocyte complex maturation………….……………………………………….44 Gonadotropins in maturation media influence maturation rate but not MIR21 expression in MII arrested oocytes……………………..…………..45 Cumulus cells influence oocyte maturation but not MIR21 expression in MII arrested oocytes…………………………………...…………………45 Inhibition of MIR21 affects oocyte maturation and PDCD4 protein expression…………………………………………………………………....57 MIR21 inhibition during in vitro maturation impacts parthenogenetic embryo development………………………………………………………………….66 MIR21 expression in the developing pig follicle…………………………….66 Discussion…………...……………………………………………………….69 Chapter 4. Heat stress during pig oocyte in vitro maturation impacts embryonic development and alters expression of molecular markers……………………………….76 Abstract………………………………………………………………………………77 Introduction……………………………………………………………………….….79 Materials and methods……………………………………………………………….81 Oocyte collection and In vitro Maturation…………………………………...81 In vitro fertilization and culture……………………………………………...82 Quantitative RT-PCR………………………………………………………...83 Statistical analysis……………………………………………………………83 v Results……………………………………………………………………………….84 Oocyte maturation and blastocyst rates are influenced by heat stress……....84 Characterization of gene expression in MII arrested oocytes and 4-cell stage embryos following in vitro maturation….………………………91 MicroRNA (MIR21) and mRNA target programmed cell death 4 (PDCD4)…………………………………………………………………….91 Heat Shock Proteins………………………………………………………….96 Discussion…………………………………………………………………………..101 Chapter 5. MiRNA during Rapid Trophoblastic Elongation of the Porcine Conceptus………………………………………………………………………….105 Abstract……………………………………………………………………………..106 Introduction…………………………………………………………………………107 Materials and Methods……………………………………………………………...109 Conceptus Collection……………………………………………………….109 RNA Extraction…………………………………………………………….109 SOLiD Sequencing ………………………………………………………...110 Analysis and Classification of Short Reads………………………………..111 Quantitative RT-PCR……………………………………………………….115 MiRNA Pathways and gene targets……………………………………….. 115 Statistical Analysis…………………………………………………………116 Results………………………………………………………………………………116 SOLiD Sequence Results………………………….……………………….116 vi MiRNA Chromosome Distribution………………………………………..119 Quantitative RT-PCR via stem-loop miRNA assay……………………….119 MiRNA Pathway prediction with DIANA-mirPath………………………124 Discussion…………………………………………………………..…………….127 Chapter 6. Summary and Conclusion……………………………………………………131 REFERENCES CITED…………………………………………………………………...133 APPENDIX I. MiRNA expression identified by Ensemble by read greatest to least for each stage of conceptus development: D12 spherical, D12 filamentous, and D14 filamentous………………………………………………………………………………….157 APPENDIX II. Protocol for in vitro production of porcine embryos…………..………...165 APPENDIX III. Western blot protocol………………………………………….………..167 APPENDIX IV. Protocol for tunnel assay……………………….……….……………….168 APPENDIX V. Immunostaining for oocytes and early embryos………………..…….…..169 APPENDIX VI. In situ hybridization of ovary sections…………………..………………170 vii LIST OF TABLES Table 3.1 Summary of oocyte development to MII arrest and 4-cell stage of embryonic development at 60 hrs following parthenogenetic activation………………………………..60 Table 5.1 MIRNA function predicted by DIANA miR-PATH……………………………125 viii LIST OF FIGURES Figure 2.1 Diagram of the 21 day estrous cycle of the pig...…………………………..…...8 Figure 2.2 MiRNA are differentially expressed between GV and MII arrested oocytes.…...31 Figure 3.1 MIR21 expression is significantly increased in the cumulus cells of MII oocytes compared to cumulus cells of GV oocytes and has an inverse relationship with PDCD4 protein in the oocyte during maturation from GV to MII………...…….…….47 Figure 3.2 PDCD4 immunostaining to detect protein expression demonstrated a decrease in PDCD4 protein during oocyte progression from GV to MII arrest....…………..50 Figure 3.3 Luteinizing hormone and follicle stimulating hormone affect oocyte maturation to MII arrest and the lack of FSH decreased MIR21 expression in MII oocytes………………………………………………………………………………..……..52 Figure 3.4 Oocyte maturation was affected by the absence of cumulus cells; however MIR21 expression was not affected by the presence or absence of cumulus cells.………...54 Figure 3.5 Antisense MIR21 FAM labeled PNA added to maturation media to demonstrate translocation of PNA during in vitro maturation into cytoplasm of cells in the cumulus oocyte complex …………………………………………………….56 ix Figure 3.6 Oocytes cultured with MIR21 inhibitor for 42 hrs during in vitro maturation..……………………………………. …………………………..……..………...59 Figure 3.7 PDCD4 protein expression between samples following analysis and quantification of band intensity..……………………………………..………………...63 Figure 3.8 PDCD4 immunostaining of oocytes following in vitro maturation……………65 Figure 3.9 In situ hybridization of MIR21 in the gilt ovaries to identify MIR21 expression during follicle development….…………………………………………………68 Figure 3.10 Proposed mechanism of MIR21 function in the developing follicle and maturing oocytes.………….…………………………………………………………………75 Figure 4.1 Maturation rates for MII development of oocytes heat stressed during IVM…...86 Figure 4.2 Percentage of IVF embryos produced from oocytes heat stressed during IVM, which developed to >4-cell stage at 60 hrs post fertilization………….……..…..……88 Figure 4.3 Percent blastocyst development from oocytes heat stressed during IVM………90 Figure 4.4 MIR21 and PDCD4 mRNA expression in heat stressed MII arrested x oocytes……………………………………………………………………………………..93 Figure 4.5 MIR21
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