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Identification and Characterization of Micrornas in Porcine Gametes and Pre-Implantation Embryos Erin Curry Clemson University, [email protected]

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Identification and Characterization of Micrornas in Porcine Gametes and Pre-Implantation Embryos Erin Curry Clemson University, Curry3@Clemson.Edu Clemson University TigerPrints All Dissertations Dissertations 12-2010 Identification and Characterization of MicroRNAs in Porcine Gametes and Pre-Implantation Embryos Erin Curry Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations Part of the Animal Sciences Commons Recommended Citation Curry, Erin, "Identification and Characterization of MicroRNAs in Porcine Gametes and Pre-Implantation Embryos" (2010). All Dissertations. 633. https://tigerprints.clemson.edu/all_dissertations/633 This Dissertation is brought to you for free and open access by the Dissertations at TigerPrints. It has been accepted for inclusion in All Dissertations by an authorized administrator of TigerPrints. For more information, please contact [email protected]. IDENTIFICATION AND CHARACTERIZATION OF MICRORNAS IN PORCINE GAMETES AND PRE-IMPLANTATION EMBRYOS A Dissertation Presented to the Graduate School of Clemson University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Animal & Veterinary Sciences by Erin Curry December 2010 Accepted by: Dr. Scott L. Pratt, Committee Chair Dr. Frank A. Feltus Dr. James C. Morris Dr. Thomas R. Scott i ABSTRACT MicroRNAs (miRNAs) are short ribonucleic acids that ultimately affect the production of proteins. Although miRNAs are involved in nearly every biological process examined to date, little is known of the identity or function of miRNA in porcine reproductive tissues or their potential involvement in reproductive processes in pigs or other species. The objective of this dissertation research was to determine the presence of miRNAs in porcine gametes and both in vivo- and in vitro- produced pre-implantation embryos and to identify differences in miRNA expression between normal and aberrant samples. Using a heterologous RT-PCR approach, we demonstrated the presence of a total of 92 miRNAs in porcine oocytes, spermatozoa, and/ or embryos at the 4-cell, 8- cell, 16-cell, and blastocyst stages, with hundreds more predicted by miRNA microarray. Subsequent qRT-PCR analysis showed differential expression of five miRNAs, let-7a, - 7d, -7e, miR-15b, and -22, between normal sperm and morphologically abnormal sperm or sperm samples exhibiting low motility. Messenger RNA targets of the differentially expressed miRNAs encode proteins important for spermatogenesis, sperm structure, and/ or sperm cell metabolism. Differential expression was also shown among embryos at various stages in development, demonstrating a temporal expression pattern of specific miRNAs in pre-implantation embryo growth. More interestingly, miR-24 was differentially expressed between in vivo- and in vitro- produced embryos at the 8-cell and blastocyst stages, supporting the need to characterize aberrant miRNA expression associated with the abnormal embryonic development correlated with assisted reproductive technologies. All of the miRNAs examined demonstrated high sequence ii similarity to the corresponding human miRNA sequences, indicative of high conservation among species. Understanding miRNA expression in reproductive processes is critical to comprehending the mechanistic roles miRNAs play in the regulation of all physiological processes. iii ACKNOWLEDGMENTS First, I thank my committee members: Dr. Scott Pratt, for giving me the opportunity to pursue a PhD in his lab and for both his guidance and absence exactly when needed; Dr. Alex Feltus, who significantly contributed to both my interest in and understanding of bioinformatics; Dr. John Gibbons, for his reliability in offering assistance at every embryo collection and for introducing me to the field of assisted reproductive technologies; Dr. Jim Morris, for dependably offering challenging, thought-provoking questions, and; Dr. Tom Scott, for providing input on methodologies and experimental design. There are many others who provided support over the past few years: the Clemson Swine Farm staff including Harold Abercrombie and Lindsay Dacus for their care of dozens of gilts; Dr. Brian Bolt for his hard work at the pig slaughters; Drs. Rebecca Krisher and Melissa Paczkowski (University of Illinois), for generating the IVF embryos; Heather Stowe, for being a considerate lab mate, for helping enormously with the animal work, and for making parthenote production days a lot shorter; Samantha Calcatera, for keeping it interesting both in work and out and for being a willing dog-sitter and drinking buddy– I couldn‟t have asked for better lab partners; Dr. Steve Ellis, for consistently providing valuable criticism on all of my writings, and; Dr. Marcy Owens for making this last semester run more smoothly than any of the previous. I also thank my Creative Inquiry students: Blake Long, Allison Hauge, Stacey Klinar, Brittany Donahue, Julie Lemons and Courtney Smith who assumed heat check and AI responsibilities, Rabun Howard, and Andrew Enders, who helped tremendously with embryo RNA isolations. iv TABLE OF CONTENTS Page TITLE PAGE .................................................................................................................... i ABSTRACT ..................................................................................................................... ii ACKNOWLEDGMENTS .............................................................................................. iv LIST OF TABLES ......................................................................................................... vii LIST OF FIGURES ...................................................................................................... viii CHAPTER I. LITERATURE REVIEW .............................................................................. 1 Overview .................................................................................................. 1 History...................................................................................................... 2 Evolution and conservation...................................................................... 3 Synthesis and function ............................................................................. 5 Regulation of miRNA expression .......................................................... 15 Roles in reproduction ............................................................................. 17 Assisted reproductive technologies........................................................ 29 Methodologies to detect miRNA ........................................................... 30 Why pigs? .............................................................................................. 37 II. INTRODUCTION ....................................................................................... 42 Objective ................................................................................................ 43 III. DETECTION OF PORCINE SPERM MICRORNAS USING A HETEROLOGOUS MICRORNA MICROARRAY AND REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION ................. 45 Introduction ............................................................................................ 45 Materials and Methods ........................................................................... 46 Results .................................................................................................... 49 Discussion .............................................................................................. 55 v Table of Contents (Continued) Page IV. DIFFERENTIAL EXPRESSION OF PORCINE SPERM MICRORNAS AND THEIR ASSOCIATION WITH SPERM MORPHOLOGY AND MOTILITY ............................................................................................ 58 Introduction ............................................................................................ 58 Materials and Methods ........................................................................... 59 Results .................................................................................................... 65 Discussion .............................................................................................. 67 V. IDENTIFICATION OF MICRORNAS IN PORCINE OOCYTES AND IN VIVO- PRODUCED EMBRYOS .......................................................... 70 Introduction ............................................................................................ 70 Materials and Methods ........................................................................... 70 Results .................................................................................................... 74 Discussion .............................................................................................. 81 VI. COMPARATIVE MICRORNA EXPRESSION IN BOTH IN VIVO- AND IN VITRO PRODUCED PRE-IMPLANTATION EMBRYOS ................. 82 Introduction ............................................................................................ 82 Materials and Methods ........................................................................... 83 Results .................................................................................................... 91 Discussion ............................................................................................ 100 VII. CONCLUSIONS........................................................................................ 104 APPENDICES ............................................................................................................. 106 A: Detectable miRNAs in boar sperm via microarray ...................................
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