Inaugural-Dissertation zur Erlangung der Doktorwürde (Dr. rer. biol. vet.) der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Analysis and comparison of dynamic mRNA expression changes in the equine and porcine endometrium during the estrous cycle von: Simone Mariella Gebhardt aus München München 2017 Aus dem Veterinärwissenschaftlichen Department der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Lehrstuhl für Molekulare Tierzucht und Biotechnologie Arbeit angefertigt unter der Leitung von: Univ.-Prof. Dr. Eckhard Wolf Mitbetreuung durch: Priv.-Doz. Dr. Stefan Bauersachs Gedruckt mit Genehmigung der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Dekan: Univ.-Prof. Dr. Reinhard K. Straubinger, PhD Berichterstatter: Univ.-Prof. Dr. Eckhard Wolf Korreferent/en: Univ.-Prof. Dr. Rolf Mansfeld Tag der Promotion: 29.07.2017 CONTENTS CONTENTS CONTENTS .......................................................................................................... I ABBREVIATIONS ............................................................................................ V I. INTRODUCTION ................................................................................. 1 Morphological changes of the equine endometrium throughout the estrous cycle .... 1 Morphological changes of the porcine endometrium throughout the estrous cycle .. 2 Uterus ......................................................................................................................... 3 Endometrium .............................................................................................................. 4 Progesterone (P4) ....................................................................................................... 5 Estradiol-17ß (E2) ...................................................................................................... 6 Estrus and early luteal phase ...................................................................................... 8 Diestrus and late luteal phase ..................................................................................... 8 Estrus and metestrus ................................................................................................... 9 Luteal phase and luteolysis ...................................................................................... 10 II. MATERIALS AND METHODS ........................................................ 12 Housing of mares ..................................................................................................... 16 Housing of sows ....................................................................................................... 16 Collection of equine endometrial biopsy samples ................................................... 17 Collection of porcine endometrial biopsy samples .................................................. 17 Equine blood samples .............................................................................................. 18 Porcine blood samples .............................................................................................. 18 I CONTENTS H&E–Staining protocol ............................................................................................ 18 Tissue composition ................................................................................................... 18 Homogenization ....................................................................................................... 19 Phase separation ....................................................................................................... 19 RNA-precipitation .................................................................................................... 19 Pooling of RNA isolated from porcine endometrial tissue samples ........................ 20 Spectrophotometrical analysis of nucleic acids ....................................................... 21 2.5.5.1. RNA ........................................................................................................................ 21 2.5.5.2. DNA ........................................................................................................................ 22 Microfluidics-based electrophoresis for sizing, quantification and quality control of nucleic acids ............................................................................................................. 22 2.5.6.1. Analysis of RNA samples ........................................................................................ 22 2.5.6.2. Analysis of DNA samples ........................................................................................ 22 Agarose gel electrophoresis ..................................................................................... 22 Agilent microarray analysis ..................................................................................... 23 2.5.8.1. Agilent Quick Amp Labeling Kit, one-color ........................................................... 23 2.5.8.2. Agilent Low RNA Input Quick Amp Labeling Kit, one-color ................................ 24 2.5.8.3. cDNA synthesis ........................................................................................................ 24 2.5.8.4. Production of Cy3-labelled cRNA ........................................................................... 24 2.5.8.5. Hybridization to microarrays ................................................................................... 25 2.5.8.6. Scanning of microarrays .......................................................................................... 25 Data analysis for microarrays ................................................................................... 26 2.5.9.1. Feature extraction, filtering, normalization and statistical analysis ......................... 26 2.5.9.2. Cluster analysis and DAVID functional annotation clustering ................................ 26 Quantitative real–time RT-PCR (qPCR) .................................................................. 27 2.5.10.1. cDNA synthesis ........................................................................................................ 27 2.5.10.2. Quantitative real-time PCR protocol ........................................................................ 27 Analysis of qPCR datasets ....................................................................................... 28 Analysis and purification of qPCR products ............................................................ 28 „Sanger“-sequencing of purified qPCR products .................................................... 28 III. RESULTS ............................................................................................. 29 Isolated RNA from equine samples ......................................................................... 32 Isolated RNA from porcine samples ........................................................................ 33 Heatmap ................................................................................................................... 35 Clustering of similar mRNA expression profiles (MeV/SOTA) ............................. 36 Functional annotation clustering .............................................................................. 39 Genes involved in prostaglandin metabolism and signaling and in steroid hormone signaling ................................................................................................................... 40 II CONTENTS Genes related to ‘regulation of apoptosis’ ............................................................... 42 Genes related to angiogenesis, blood circulation and vasculature development ..... 43 Heatmap ................................................................................................................... 44 Clustering of similar mRNA expression profiles (MeV/SOTA) ............................. 45 Functional annotation clustering .............................................................................. 47 Genes related to prostaglandin metabolism and signaling and to steroid hormone signaling ................................................................................................................... 48 Genes related to ‘regulation of apoptosis’ ............................................................... 50 Genes related to angiogenesis, blood circulation and vasculature development ..... 51 Analysis of selected genes ( Equus caballus ) ........................................................... 53 Analysis of selected genes ( Sus scrofa ) ................................................................... 55 Relation of positively correlated genes to SOTA clustering analyses ..................... 57 Relation of negatively correlated genes to SOTA clustering analyses .................... 58 Analysis of positive correlated genes ....................................................................... 59 Analysis of negatively correlated genes ................................................................... 61 Comparison to dynamic gene expression changes during the estrous cycle in bovine endometrium (Mitko, 2008) ..................................................................................... 62 4.2.3.1. Positively correlated genes ....................................................................................... 62 4.2.3.2. Negatively correlated genes ..................................................................................... 62 IV. DISCUSSION ......................................................................................