Effect of Maternal Age on Transcriptome of Granulosa Cells from Bovine Dominant Follicles A Thesis submitted to the College of Graduate Studies and Research In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy In the Department of Veterinary Biomedical Sciences University of Saskatchewan Saskatoon By MUHAMMAD IRFAN-UR-REHMAN KHAN © Copyright Muhammad Irfan-ur-Rehman Khan, January, 2014. All rights reserved. PERMISSION TO USE In presenting this thesis in partial fulfillment of the requirements for the degree of Doctor of Philosophy from the University of Saskatchewan, I agree that the Libraries of this University may take it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Request for permission to copy or to make other use of material in this thesis in whole or part should be addressed to: Head of the Department of Veterinary Biomedical Sciences Western College of Veterinary Medicine 52 Campus Drive, University of Saskatchewan Saskatoon, Saskatchewan, S7N 5B4 Canada i ABSTRACT Advanced maternal age has been shown to influence follicular and luteal dynamics in bovine ovary resulting in reduced fertility. The overall objective of the four studies presented in this thesis is to identify the maternal age-associated transcriptional changes in granulosa cells of the dominant follicles during follicle development. In the first study, mRNA expression levels of housekeeping genes were measured by real–time quantitative PCR (RT-qPCR) in granulosa cells of dominant follicles and FSH- stimulated follicles to select and validate suitable reference genes for relative gene expression analyses during maternal and follicular aging. Stability of six reference genes (GAPDH, ACTB, EIF2B2, UBE2D2, SF3A1 and RNF20) was analyzed using GeNorm, DeltaCT and NormFinder programs and comprehensive ranking order was determined based on these programs. Geometric mean of multiple genes (UBE2D2, EIF2B2, GAPDH and SF3A1) was more appropriate reference control than individual genes for the comparison of relative gene expression among dominant and FSH-stimulated follicles during maternal and/or follicular aging studies. In the second study, maternal age-associated changes in the transcriptome of granulosa cells recovered at the time of selection of the dominant follicle from aged (n=3) and young cows (n=3) were determined by EmbryoGENE bovine oligo-microarrays (EMBV3, Agilent Technology). The mRNA expression of five transcripts (CYP19A1, PCNA, GJA1, TPM2, and VNN1) was confirmed in a different set of granulosa cell samples by RT-qPCR to validate microarray data. A total of 169 genes/isoforms were differentially expressed (≥ 2-fold-change; P ≤ 0.05) in aged cows vs. young cows. These transcripts revealed inefficient 1) control of gonadotropins, and gonadotropin-induced changes in the cytoskeleton and extracellular matrix, ii 2) lipid metabolism and steroidogenesis 3) cell proliferation, cell cycle control and intercellular communication, and 4) higher oxidative stress responses in aged cows vs. young cows. In the third study, changes in the transcriptome of granulosa cells of the preovulatory follicle 24 h after LH treatment from aged (n= 3) and young (n=3) were determined. A total of 1340 genes were expressed differentially (≥ 2-fold change; P ≤ 0.05) in aged cows vs. young cows. The mRNA expression of five transcripts (RGS2, PTGS2, TNFAIP6, VNN1, NR5A2 and GADD45B) was confirmed in a different set of granulosa cell samples to validate microarray data. These transcripts were related to delayed 1) response to LH treatment 2) cellular differentiation and luteinization and 3) progesterone synthesis. Intra-follicle levels of progesterone were lower (P < 0.05) in aged cows compared to young and mid-aged cows. The fourth study compared the aged-associated changes in the transcriptome of granulosa cells during follicle development from the time of dominant follicle selection to preovulatory stage (24 h after LH). In comparison to young cows, aged cows expressed fewer differentially expressed genes/isoforms (1206 vs. 2260, respectively) at ≥ 2-fold-change (P ≤ 0.05) in the granulosa cells of the preovulatory (24 h after LH treatment) vs. the dominant follicle at selection. These transcripts in aged cows were related to late and inefficient 1) organization of cytoskeleton and cytoplasm, 2) differentiation, 3) lipid and cholesterol metabolism, 4) proliferation and 5) higher response to oxidative stress and free radical scavenging in the preovulatory follicles vs. the dominant follicle at selection. In conclusion, maternal age-alters the gene expression of granulosa cells of the dominant follicles during follicle development and results in a compromised follicular environment. iii ACKNOWLEDGEMENT First, I thank you, Dr. Jaswant Singh for accepting me in your lab and teaching me how to think out of the box and for encouraging me to learn Microarrays, Bioinformatics, Statistics, and Scientific writing. You never hesitated to sit beside your students in classrooms nor did you stop asking questions to help them understand the science. I recognize your time, patience and effort to help me achieve the standards. Second, I am thankful to my Ph.D. advisory committee. Dr. Marc-Andre Sirard: for helping me to excel in analyzing the complex biological context of Microarray data. Dr. Gregg Patrick Adams: Thank you for the critical and encouraging remarks that helped me to polish my abilities. I will always remember the time spent alongside you while teaching Veterinary Anatomy to the D.V.M. students and during colpotomy sessions at the barn. Dr. Muhammad Anzar: I owe you so much that I wish could return the support and efforts that you and your family extended to me and my wife to make my stay comfortable in Saskatoon. Dr. Vikram Misra: Thank you for helping me with qPCR. Dr. Gillian Muir (Graduate Chair): I appreciate your guidance and valuable comments during my stay at department. Dr. Ruben Mapletoft: Thank you for having me in thought-provoking discussions with you during my research. I also recognize the help of Isabelle Dufort (Lab assistant EmbryoGene Network) to carryout qPCR and microarray hybridization at the University of Laval. Third, I am thankful to comrade graduate students and their families for making Saskatoon my home. I regard Attiq Hassan and his family for spending wonderful time with my family in Saskatoon. I also owe sincere thanks to Fernanda, Dinesh, Miriam, Manuel, Paula, Marco, Andreas, Hayder, Parkhe, Ajeet and Orleigh for helping me to scan cows at Goodale farm and for extending the favors. I also extend my appreciation to undergraduate summer iv students Kelly, Amber, Tegan, and Dali for being an excellent support during my work at Goodale. I have a lot of respects for Susan; Sandra and Cheryl (Secretaries at Graduate office), Khosala, Loyal Boswal, (Laboratory Research Assistants) for always being cooperative. Fourth, I cherish my school, college, and university teachers (UVAS): Sir, Sarwar Bhatti, Sir Nazar Bhatti, Sir Lateef Bhatti, and Sir Naeem Akbar, Dr. Nasim Ahmad and Dr. Ijaz Ahmad and Dr. Aleem Bhatti, who adorned me with wings of knowledge. I also extend my heartiest thanks to the Higher Education Commission of Pakistan and the University of Veterinary and Animal Sciences, Lahore for providing me scholarship to pursue my PhD at University of Saskatchewan. I look forward to serving you my Alma mater (UVAS) with all of my abilities and enthusiasm. Lastly, I thank my wife Mehreen for her companionship, support and for bringing me the joys (Rayyan and Miraan). I love you and realize the hardships you bore for me. My Mother and Father, thank you for nurturing me to be a good person, I lived up to this day because of your prayers. May ALLAH bless both of you long life and health (Amin). v DEDICATION I dedicate this thesis to my wife and parents for their unconditional love and support for me. vi TABLE OF CONTENTS PERMISSION TO USE ................................................................................................................. i ABSTRACT ................................................................................................................................... ii ACKNOWLEDGEMENT ........................................................................................................... iv DEDICATION.............................................................................................................................. vi TABLE OF CONTENTS ........................................................................................................... vii LIST OF TABLES ....................................................................................................................... xi LIST OF ABBREVIATIONS ................................................................................................. xviii CHAPTER 1: GENERAL INTRODUCTION ..........................................................................