THE EFFECT OF EXPRESSION OF ESTRUS AT ARTIFICIAL INSEMINATION ON TARGET GENES IN THE ENDOMETRIUM, CONCEPTUS AND CORPUS LUTEUM OF BEEF COWS by Saeideh Davoodi A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate and Postdoctoral Studies (Applied Animal Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2015 © Saeideh Davoodi, 2015 Abstract The aim of this study was to test the effect of expression of estrus at artificial insemination (AI) on the endometrium, conceptus and corpus luteum (CL) gene expression. Twenty-three multiparous non-lactating Nelore cows were enrolled on an estradiol (E2) and progesterone (P4) based timed-AI protocol (AI = d 0), then slaughtered for endometrium, CL and conceptus collection on d 19. Body condition score (BCS), blood samples and ultrasound examination was performed on d 0, 7 and 18 of the experiment followed by RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of 58 target genes. Data was checked for normality and analysed by ANOVA for repeated measures using proc GLM, MIXED and UNIVARIATE. Estrous expression had no correlation with parameters such as BCS, pre-ovulatory follicle and CL diameter, P4 concentration in plasma on d 7 and 18 after AI and IFN-tau concentration in the uterine flushing (P > 0.05); however, a significant increase was observed in conceptus size (P = 0.02; 38.3 ± 2.8 vs 28.2 ± 2.9). The majority of transcripts affected by estrous expression in the endometrium belong to the immune system and adhesion molecule family (MX1, MX2, MYL12A, MMP19, CXCL10, IGLL1 and SLPI) (P ≤ 0.05). Genes related to apoptosis, P4 synthesis and prostaglandin receptor were down-regulated (CYP11A, BAX and PGF2α receptor) (P < 0.05) in the CL tissue of cows in estrus. In addition, four genes were identified as differentially expressed in the 19 old conceptus from cows observed in estrous (ISG15, PLAU, BMP15 and EEF1A1; P<0.05). There was also a significant effect of P4/d7 mainly affecting immune system, adhesion molecules and wnt signaling pathway of endometrium (IGLL1, MX2, SLPI, TRD, APC, WNT2, GLYCAM1 and MYL12A) (P<0.05). A significant interaction between estrus expression and P4 concentration on d 7 was more pronounced in immune system genes (MX1, MX2, TRD, SLPI and IGLL1; P<0.05). This study demonstrated that estrous expression at the time of AI, in spite of ovulation being ii induced by E2, could alter the gene expression profile in reproductive tissues during the pre- implantation phase. iii Preface The animal part of the study and sample collection was conducted from January to March (Summer) at a commercial cow–calf operation located in Mineiros, Goias, Brazil and was part of the study in the university of Sauo Paulo. The animals used herein were cared for in accordance with the practices outlined in the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (Curtis and Nimz, 2010). Animal Care (CCAC) / National Institutional Animal User Training (NIAUT) Program was obtained under this certificate #6570-14. I was responsible for gene expression studies ( performed at the University of British Columbia’s Dairy Education and Research Centre in Agassiz, BC) and at Dr. Suzanne Clee’s laboratory for Diabetes Research Group at the Life Science Centre, as wells as data analysis. iv Table of Contents Abstract ......................................................................................................................................................... ii Preface ......................................................................................................................................................... iv Table of Contents .......................................................................................................................................... v List of Tables ............................................................................................................................................... vii List of Figures ............................................................................................................................................. viii List of Abbreviations .................................................................................................................................... ix Acknowledgements .....................................................................................................................................xiii Dedication ................................................................................................................................................... xiv 1 Literature review ...................................................................................................................................... 1 1.1 Overview of subfertility in cattle ................................................................................................ 1 1.2 Estrus cycle .................................................................................................................................. 3 1.3 Pregnancy establishment ............................................................................................................ 5 1.3.1 The role of the corpus luteum during pregnancy ............................................................... 5 1.3.2 Endometrium remodelling and embryo development ....................................................... 7 1.4 Role of steroids during pregnancy establishment ...................................................................... 9 1.4.1 Progesterone function ........................................................................................................ 9 1.4.2 Estradiol function .............................................................................................................. 10 1.5 Effect of proestrus length on pregnancy maintenance ............................................................ 14 1.6 Interferon, pregnancy stimulated genes in reproductive tissue .............................................. 15 1.6.1 Genes involved in endometrium receptivity ..................................................................... 16 1.6.2 Apoptosis, steroid synthesis and angiogenesis in CL ........................................................ 38 1.6.3 Developmental and immune related genes in the conceptus .......................................... 42 2 Impact of estrus exposure on gene expression of the endometrium, corpus luteum and conceptus of beef cows ................................................................................................................................................ 50 2.1 Hypothesis and objectives of the dissertation .......................................................................... 50 2.2 Material and methods .............................................................................................................. 50 2.2.1 Animals, housing and grouping ......................................................................................... 51 2.2.2 Blood samples and ultrasound examinations ................................................................... 52 2.2.3 Slaughter and tissue collection ......................................................................................... 52 2.2.4 RNA extraction .................................................................................................................. 53 2.2.5 Primer design .................................................................................................................... 53 2.2.6 Complementary DNA synthesis ......................................................................................... 53 2.2.7 Quantitative real time PCR ................................................................................................ 54 v 2.2.8 Statistical analysis.............................................................................................................. 55 2.3 Results ....................................................................................................................................... 55 2.3.1 Expression of genes in the endometrium ......................................................................... 55 2.3.2 Expression of genes in the corpus luteum affected by estrus expression ........................ 56 2.3.3 Gene expression in the embryo ........................................................................................ 57 2.3.4 Ovarian and embryo parameters ...................................................................................... 57 2.3.5 Effect of P4 concentration on day 7 on animal variables, embryo genes, endometrium gene expression and estrus outcome ................................................................................................. 57 2.3.6 Effect of embryo size on animal variables and embryo gene expression ......................... 58 2.4 Discussion.................................................................................................................................. 58 3 General discussion ............................................................................................................................ 82 3.1 Summary ................................................................................................................................... 82 3.2 Limitations ...............................................................................................................................