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The Pennsylvania State University the Graduate School College of Agricultural Sciences The Pennsylvania State University The Graduate School College of Agricultural Sciences EXPRESSION OF MICRONRNAS IN THE BOVINE CORPUS LUTEUM AND THEIR IMPACT ON LUTEAL CELL FUNCTION A Thesis in Animal Science by Courtney Lynne Smith © 2010 Courtney Lynne Smith Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science August 2010 The thesis of Courtney Lynne Smith was reviewed and approved* by the following: Joy L. Pate Professor of Reproductive Physiology C. Lee Rumberger and Family Chair in Agricultural Sciences Thesis Advisor Wansheng Liu Associate Professor of Genomics Francisco J. Diaz Assistant Professor of Reproductive Biology Daniel R. Hagen Professor of Animal Science Coordinator of Graduate Programs in Animal Science *Signatures are on file in the Graduate School. ii ABSTRACT The corpus luteum is a temporary endocrine structure that produces progesterone for the maintenance of pregnancy and has many regulatory processes that are involved in its development, function, and regression. MicroRNAs are noncoding RNAs that are 17-25nt in length and function in translational repression by binding to the 3’ untranslated region of its target mRNA. Because of this repression, microRNAs are able to regulate the expression of many different genes. Drosha, an RNase III enzyme needed for processing the primary microRNA transcript into the precursor microRNA form, is considered one of the most important components in microRNA biogenesis. Little is known about microRNA expression and regulation pertaining in the corpus luteum. Even less is known about miRNA expression in livestock species. The studies presented here are the first studies focusing on microRNA expression in the bovine corpus luteum and miRNA regulatory abilities in bovine luteal cells. To investigate the expression of different miRNAs in luteal tissue, corpora lutea were removed from cyclic Holstein cows on days 4, 10, and 18 of the estrous cycle, and on day 18 of pregnancy. Total RNA enriched with small RNAs were isolated from each treatment and were hybridized to Affymetrix microRNA microarrays. Quantitative PCR was used to confirm microarray results. To assess the importance of microRNAs to luteal cell function, luteal cells were collected after the dissociation of midcycle (day 10-11) luteal tissues and luteal cells were transfected with siRNAs against Drosha to inhibit microRNA biogenesis. Transfected cells were treated with or without LH or TNF-α and IFN-γ to assess the effect of decreased microRNA expression on progesterone production and apoptosis, respectively. iii Bovine and nonbovine specific microRNAs were found to be differentially expressed among luteal tissue collected on day 4, 10, and 18 of the estrous cycle and on day 18 of pregnancy. Three microRNAs, miR-10a, miR-103, and miR-99a were confirmed to have increased expression in luteal tissue on day 18 of pregnancy. Overall, thirteen nonbovine miRNAs not previously reported as present in bovine tissues were detected only in luteal tissue on day 18 compared to three miRNAs expressed only in luteal tissue on day 18 of pregnancy. Nonbovine microRNAs were also identified in bovine luteal tissue on day 10 and day 4, with 42 miRNAs expressed only on day 10 and thirteen miRNAs expressed only on day 4. The knockdown of Drosha mRNA expression in luteal cells cultured with LH resulted in a significant decrease (P ˂ 0.05) in progesterone production compared to luteal cells transfected with Drosha siRNAs cultured with no LH. Also, the percentage of apoptosis for cells transfected with Drosha siRNAs and treated with or without TNF-α and IFN-γ significantly increased (P ˂ 0.05) compared to the controls. Overall, these data confirm the presence and differential expression of miRNAs in luteal tissue during the luteal phase and suggest that microRNAs have a role in luteal cell function and survival. iv TABLE OF CONTENTS ABSTRACT……………………………………………………………………………………………iii LIST OF TABLES …………………………………….………………………………………............vii LIST OF FIGURES…………………………..………….……………………………………………..ix ACKNOWLEDGEMENTS……………………………..……………………………………………..x DEDICATION…………………………………………………………………………………………..xiii CHAPTER 1: REVIEW OF LITERATURE……………………………………......1 1.1 Luteinization……………………………………………………………........1 Hormonal Initiation of Luteinization………………………………..1 Morphological and Cellular Changes………………………….......2 Steroidogenic Changes…………………………………………......2 Corpus Luteum Size Increase……………………………………....3 Corpus Luteum Angiogenesis………………………………….......4 1.2 Luteotropism………………………………………………………………...5 Cellular Content of the Corpus Luteum……….……………………5 Progesterone Production…………………………………………….7 Other Factors Involved in Progesterone Production…………….10 1.3 Luteolysis…………………………………………………………………...11 Prostaglandin F2α…………………………………………………...11 Prostaglandin F2α Effects on Luteal Tissue and Function……...12 1.4 Discovery of MicroRNA………………………………………………….13 1.5 MicroRNA Biogenesis…………………………………………………....15 MicroRNA Gene Transcription……………………………………15 Nuclear Processing………………………………………………..15 Cytoplasmic Processing……………………………………..…….17 1.6 Assembly of MicroRNAs into Effector Complexes………………...18 1.7 MicroRNA Target Recognition…………………………………………18 v 1.8 MicroRNA Function……………………………………………………...20 1.9 MicroRNA Expression in the Ovary..................................................21 MicroRNA Expression in Bovine Tissues……………………...24 1.10 Statement of Problem…………..……………………………………..27 1.11 Hypothesis and Implications…………………………………….…..28 CHAPTER 2: EXPRESSION OF MICRORNAS IN THE BOVINE CORPUS LUTEUM……………………….…………………………………...29 Introduction………………………………………………………………..29 Materials and Methods…………………………………………………..31 Results……………………………………………………………………..34 Discussion………………………………………………………………….39 CHAPTER 3: THE ROLE OF DROSHA IN LUTEAL CELL FUNCTION………………..…………….……………………………………...50 Introduction………………………………………………………………..50 Materials and Methods…………………………………………………..52 Results……………………………………………………………………..58 Discussion………………………………………………………………...60 FINAL CONCLUSONS………………………………………………………66 FIGURES…………………………………………………………………........68 REFERENCES……………………………………………………………....110 vi LIST OF TABLES Table Page Table 1: Bovine-specific microRNAs differentially expressed in luteal tissue from day 10 cyclic versus day 4 cyclic……………………………………………………………………………………71 Table 2: MicroRNAs present in day 18 bovine lutea tissue detected with heterlogous probes………………………………………………………………………………………………….75 Table 3: MicroRNAs present in day 10 and day 4 bovine luteal tissue detected with heterologous probes………………………………………………………………………………….77 Table 4: MicroRNAs in bovine luteal tissues in day 18 cyclic cattle not known in the cow but known microRNAs expressed in human, mouse, rat, or rhesus monkey ………………………83 Table 5: MicroRNAs in bovine luteal tissues in day 18 pregnant cattle not known in the cow but known microRNAs expression in human, mouse, rat, or rhesus monkey………………………84 Table 6: MicroRNAs in bovine luteal tissues in day 10 cyclic cattle not known in the cow but known microRNAs expressed in human, mouse, rat, or rhesus monkey……………………….84 Table 7: MicroRNAs in in bovine luteal tissues in day 4 cyclic cattle not known in the cow but known microRNAs expressed in human, mouse, rat, or rhesus monkey……………………….87 Table 8: Predicted targets for bovine specific microRNAs expressed in luteal tissue on day 18 of pregnancy…………………………………………………………………………………………...88 Table 9: Selected targets for miRNAs not yet described in the cow, but expressed in day 18 luteal tissue based on heterologous miRNA expression………………………………………………………………………………………………88 vii Table 10: Selected targets for miRNAs not yet described in the cow but expressed in day 10 and day 4 luteal tissues based on heterologous miRNA expression…………..………………………………………………..……………………….……….90 Table 11: Selected predicted targets for microRNAs present in bovine luteal tissue ………...94 viii LIST OF FIGURES Figure Page Figure 1: Examination of bovine-specific microRNAs in luteal tissue…………………………68 Figure 2: Bovine-specific microRNAs differentially expressed in luteal tissue from day 18 cyclic versus pregnant cattle………………………………………………………………………………69 Figure 3: Verification of differential expression of bovine-specific microRNAs at day 18 of pregnancy versus day 18 cyclic cattle using quantitative PCR analysis……………………...70 Figure 4: Distribution of species-specific microRNA capture probes resulting in the detection of luteal microRNAs which have not been identified in the cow……………………………….…104 Figure 5: Dicer and Drosha expression and siRNA mediated Dicer and Drosha knockdown in luteal cells……………………………………………………………………………………………105 Figure 6: Progesterone production of Drosha knockdown luteal cells………………………...107 Figure 7: Cytotoxic effect tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) on Drosha knockdown luteal cells……………………………………………………………………………..108 ix ACKNOWLEDGEMENTS First, I would like to thank Dr. Joy L. Pate for the opportunity to work in her lab and giving me the opportunity to work on this project. After everything that I have learn, I feel that microRNA research will be one of the most important scientific mysteries to unravel in all tissues and developmental processes, and I feel grateful to have taken part i starting the story of microRNA expression and function in the bovine corpus luteum. I would like to thank my committee members, Drs. Washeng Liu and Francisco Diaz, for their time, helpful suggestions and critiques, and encouragement. The Pate and Ott labs are constantly intertwined, whether in lab meetings or in scientific troubleshooting. I would
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