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Implantation Period Using the Affymetrix Genechip Mirna 4.0 Array

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Characterization of miRNAs in the spent media throughout the pre- implantation period using the Affymetrix Genechip miRNA 4.0 array by Paul Del Rio A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Biomedical Science Guelph, Ontario, Canada © Paul Del Rio, January, 2021 ABSTRACT CHARACTERIZATION OF MIRNAS IN THE SPENT MEDIA THROUGHOUT THE PRE- IMPLANTATION PERIOD USING THE AFFYMETRIX GENECHIP MIRNA 4.0 ARRAY Paul Del Rio Advisor(s): University of Guelph, 2021 Dr. Pavneesh Madan Distinct miRNA populations have only been detected in spent media (SM) cultured with blastocyst-stage embryos. Therefore, the aim of the study was to globally profile the extracellular microRNA (miRNA) population throughout the pre-implantation period in bovine embryos. Briefly, embryos were in-vitro produced in group culture and SM was collected at the 2-cell, 8-cell, and blastocyst stage of development. Total RNA was extracted from SM and hybridized onto a miRNA microarray. Results show that distinct miRNA populations can be detected in the SM at the 2-cell, 8-cell, and blastocyst stage of development with miRNAs showing stage-dependant and stage-independent expression in SM. In addition, distinct miRNA populations can be identified in the SM cultured with slow-growing and fast-growing embryos at the 2-cell, 8-cell, and blastocyst stage of development. Overall, the findings contribute to the growing body of evidence supporting the use of miRNAs in SM as non-invasive biomarkers of early embryo development. DECLARATION OF WORK PERFORMED I declare that all work presented in this thesis was performed by me with the exception of the procedures mentioned below: Stephan Botha, Saeid Ghiasi, and Stephanie Hookey collected cattle ovaries from a government inspected commercial abattoir (Cargill Meat Solutions, Guelph, Ontario, Canada). Elizabeth St. John prepared media used for in vitro production of bovine embryos. Edgardo Reyes and Allison MacKay were responsible for all the ordering of materials. Valérie Catudal from Genome Quebec (Montreal, Quebec) conducted all of the hybridization of total RNA samples on the Affymetrix Genechip miRNA 4.0 array. iii ACKNOWLEDGEMENTS First and foremost, I would like to express my deepest gratitude to my advisor, Dr. Pavneesh Madan, for giving me an opportunity to work in the field of reproductive biology. Your mentorship, guidance, and constructive feedback has allowed me to grow as an individual, student, and researcher. Giving me an opportunity to explore and choose the project of my choice has allowed me to solidify my interest and passion in this field. I will always cherish your expertise and knowledge, which has helped me through the highs and lows of my graduate school journey. I would also like to thank Dr. Jonathan Lamarre and Dr. Julang Li for sitting on my advisory committee and providing support and assistance throughout my project. I would also like to thank Dr. Monica Antenos for all her helpful advice and assistance during my project. Her unrivalled understanding of laboratory techniques allowed me to form a deeper appreciation for the process of scientific research. I would like to express my deepest appreciation for Elizabeth St. John who helped train and develop my skills in IVF. Thank you for always being there for advice and support during the beginning of my project. I would also like to thank Ed Reyes and Allison Mackay for ordering all the materials and handling the logistics involved with sending my samples to Genome Quebec. Your efficiency and organization were instrumental to the completion of my project. I would also like to thank the past and current members of the Madan lab for all your friendship and support. Thank you so much to Jyoti and Saba who were always there to lend a helping hand. I would also like to thank all the other past and present members and students in the RHBL who made my graduate school journey an enjoyable and memorable experience. iv Lastly, I would like to express my deepest appreciation to my parents and my girlfriend, Mimi, who have stood by me throughout the challenges and successes I encountered throughout my time as a graduate student. Your support, advice, and words of encouragement is source of inspiration that lifted my spirits and pushed me through difficult times. Although my dad will not be able to see me graduate, I know that he is watching from above and is proud of the person that I have become. v TABLE OF CONTENTS ABSTRACT…………………………………………………………………………………...…ii DECLARATION OF WORK PERFORMED……………………………………………...…iii ACKNOWLEDGEMENT………………………………………………………………...……iv LIST OF TABLES……………………………………………………………………………..viii LIST OF FIGURES……………………………………………………………………………..ix LIST OF ABBREVIATIONS…………………………………………………………………...x INTRODUCTION……………………………………………………………………………….1 REVIEW OF LITERATURE…………………………………………………………….……..3 Historical perspective on assisted reproductive technologies…………………………....3 Limitation of embryo transfer………………………………………………..…...………5 Embryo assessment methods………………………………………………….…..……...6 miRNAs in the spent in-vitro culture media...…..…………………………..………….14 RATIONALE……………………………………………………………………………...……25 HYPOTHESIS AND OBJECTIVES…………………………………………………….……26 CHAPTER ONE………………………………………………………………….…………….27 INTRODUCTION………………………………………………………………….…..28 MATERIALS AND METHODS………………………………………………………31 Oocyte collection and in-vitro production of bovine embryos………………….31 Collection of spent in-vitro culture media………………………………………33 miRNA extraction……………………………………………………………….33 miRNA microarray hybridization……………………….………………………34 Statistical Analysis………………………………………………………………34 Target pathway prediction of differentially expressed miRNAs…….…………35 RESULTS……………………………………………………………………………….35 Differentially expressed miRNAs in 2-cell, 8-cell and blastocyst SM………….35 Differentially expressed miRNAs shared between 2 or SM conditions……......37 vi Prediction of miRNA-mRNA targets for stage-specific and shared differentially expressed miRNAs.………………………………………………………………39 Annotated roles of differentially expressed miRNAs in literature………….….41 DISCUSSION…………………………………………………………………………...43 CHAPTER TWO……………………………………………………………………………….50 INTRODUCTION……………………………………………………………………...51 MATERIALS AND METHODS………………………………………………………54 Oocyte collection and in-vitro production of bovine embryos………………….54 Collection of spent in-vitro culture media conditioned with SG and FG embryos…………………………………………………………………………..55 miRNA extraction…………………………………………...…………….…….57 miRNA microarray hybridization……………………………………………….57 Statistical analysis……………………………………………………………….58 Target pathway prediction of differentially expressed miRNAs..………………58 RESULTS ………………………………………………………………………………59 Differentially expressed miRNAs between 2-cell SG vs. 2-cell FG, 8-cell SG vs. 8-cell FG, and blastocyst SG vs. blastocyst FG SM………………………….....59 Predictions of miRNA-mRNA targets for differentially expressed miRNAs detected between 2-cell SG vs. 2-cell FG, 8-cell SG vs. 8-cell FG, and blastocyst SG vs. blastocyst FG SM……………………………………………………..….61 DISCUSSION…………………………………………………………………………...63 SUMMARY AND FUTURE DIRECTIONS………………………………………………….68 LITERATURE CITED………………………………………………………………………...71 APPENDIX……………………………………………………………………………………...82 vii LIST OF TABLES Table 1: Spent IVC media collection schedule at various stages (adapted from Van Soom et al., 1997; Perkel and Madan, 2017). Table 2. miRNA-mRNA targets predicted to have roles in the top 3 biological pathways represented in GSEA analysis. Table 3. List of miRNAs detected in all 3 SM conditions previously annotated in literature. The majority (18 miRNAs) have been profiled in embryo-based studies, while 14 miRNAs were explored in cancer-related studies. Five miRNAs were cited in both embryo and cancer related studies. Table 4. DEM between 2-Cell SG SM vs. 2-Cell FG SM. The majority of miRNAs were upregulated in 2-cell SG SM in comparison to 2-Cell FG SM. Table 5. DEM between 8-Cell SG SM vs. 8-Cell FG SM. The majority of miRNAs were upregulated in 8-cell SG SM in comparison to 8-Cell FG SM. Table 6. DEM between blastocyst SG SM vs. blastocyst FG SM. The majority of miRNAs were upregulated in SG SM in comparison to FG SM. Table 7. Top 5 enriched biological pathways of predicted genes regulated by miRNAs differentially expressed in 2-cell SG SM vs. 2-Cell FG SM Table 8. Top 5 enriched biological pathways of predicted genes regulated by miRNAs differentially expressed in 8-cell SG SM vs. 8-Cell FG SM. Table 9. Top 5 enriched biological pathways of predicted genes regulated by miRNAs differentially expressed in blastocyst SG SM vs. blastocyst FG SM. viii LIST OF FIGURES Figure 1. Total transferable IVP and IVD embryos from the year 2000-2018. ET have been steadily increasing throughout the 18-year period, with IVP embryo surpassing IVD in 2015 as the dominate source of embryos transferred. Figure 2. A total of 111 miRNAs were differentially expressed in 2-cell, 8-cell, and blastocyst SM. Expression of miRNAs in SM increased throughout the 3 conditions examined, with 13, 21, and 77 miRNAs being detected in 2-cell, 8-cell, and blastocyst SM, respectively. The majority of miRNAs detected in SM were blastocyst derived. Figure 3. Venn diagram of DEM from 2-cell (yellow), 8-cell (green), and blastocyst SM (blue). Overlapping results showed DEM shared between 2 or more groups (14 miRNAs between 8-cell and blastocyst SM; 7 miRNAs between all 3 SM conditions). Figure 4. A total of 6 miRNAs were significantly expressed in 2-cell SM. Two miRNAs
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  • Table 1 the Statistical Metrics for Key Differentially Expressed Genes (Degs)

    Table 1 the Statistical Metrics for Key Differentially Expressed Genes (Degs)

    Table 1 The statistical metrics for key differentially expressed genes (DEGs) Agiliant Id Gene Symbol logFC pValue FDR tvalue Regulation Gene Name PREDICTED: Homo sapiens similar to Filamin-C (Gamma- filamin) (Filamin-2) (Protein FLNc) (Actin-binding-like protein) (ABP-L) (ABP-280-like protein) (LOC649056), mRNA A_24_P237896 LOC649056 0.509843 9.18E-14 4.54E-11 12.07302 Up [XR_018580] Homo sapiens programmed cell death 10 (PDCD10), transcript A_23_P18325 PDCD10 0.243111 2.8E-12 9.24E-10 10.62808 Up variant 1, mRNA [NM_007217] Homo sapiens Rho GTPase activating protein 27 (ARHGAP27), A_23_P141335 ARHGAP27 0.492709 3.97E-12 1.22E-09 10.48571 Up mRNA [NM_199282] Homo sapiens tubby homolog (mouse) (TUB), transcript variant A_23_P53110 TUB 0.528219 1.77E-11 4.56E-09 9.891033 Up 1, mRNA [NM_003320] Homo sapiens MyoD family inhibitor (MDFI), mRNA A_23_P42168 MDFI 0.314474 1.81E-10 3.74E-08 8.998697 Up [NM_005586] PREDICTED: Homo sapiens hypothetical LOC644701 A_32_P56890 LOC644701 0.444703 3.6E-10 7.09E-08 8.743973 Up (LOC644701), mRNA [XM_932316] A_32_P167111 A_32_P167111 0.873588 7.41E-10 1.4E-07 8.47781 Up Unknown Homo sapiens zinc finger protein 784 (ZNF784), mRNA A_24_P221424 ZNF784 0.686781 9.18E-10 1.68E-07 8.399687 Up [NM_203374] Homo sapiens lin-28 homolog (C. elegans) (LIN28), mRNA A_23_P74895 LIN28 0.218876 1.27E-09 2.24E-07 8.282224 Up [NM_024674] Homo sapiens ribosomal protein L5 (RPL5), mRNA A_23_P12140 RPL5 0.247598 1.81E-09 3.11E-07 8.154317 Up [NM_000969] Homo sapiens cDNA FLJ43841 fis, clone TESTI4006137.