Institute of Molecular Animal Breeding and Biotechnology Faculty of Veterinary Medicine of the Ludwig-Maximillians-University Munich Prof. Dr. E. Wolf Identification of genes induced by the conceptus in the bovine endometrium during the pre-implantation period Thesis for the attainment of the title Doctor in Veterinary Medicine from the Faculty of Veterinary Medicine of the Ludwig-Maximillians University Munich by Claudia Klein from Wiesbaden Munich 2006 Aus dem Institut für Tierzucht der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Lehrstuhl für Molekulare Tierzucht und Biotechnologie (Vorstand: Prof. Dr. E.Wolf) Identifizierung von Genen im bovinen Endometrium, welche durch den Konzeptus im prä-implantatorischen Zeitraum induziert werden Inaugural Dissertation zur Erlangung der tiermedizinischen Doktorwürde der Tierärztlichen Fakultät der Ludwig-Maximillians Universität München von Claudia Klein aus Wiesbaden München 2006 Gedruckt mit Genehmigung der Tierärztlichen Fakultät der Ludwig-Maximilians-Universtät München Dekan: Univ.-Prof.Dr. E.Märtlbauer Referent: Univ.-Prof. Dr. E. Wolf 1. Korreferent: Univ.-Prof. Dr. J. Braun 2. Korreferent: Univ.-Prof. Dr. K. Pfister 3. Korreferent: Priv.-Doz. Dr. B. Schalch 4. Korreferent: Univ. Prof. Dr. W. Schmahl Tag der Promotion: 10. Februar 2006 The following manuscript, containing the results of the present thesis, has been published in the journal “Biology of Reproduction”: Claudia Klein, Stefan Bauersachs, Susanne E. Ulbrich, Ralf Einspanier, Heinrich H.D. Meyer, Susanne E.M. Schmidt, Horst-Dieter Reichenbach, Margarete Vermehren, Fred Sinowatz, Helmut Blum, and Eckhard Wolf Monozygotic twin model reveals novel embryo-induced transcriptome changes of bovine endometrium in the pre-attachment period Biol Reprod. 2006 Feb;74(2):253-64. Epub 2005 Oct 5. Table of contents Table of contents List of figures .............................................................................................................. 1 List of abbreviations.................................................................................................... 3 1. Introduction.............................................................................5 2. Review of the literature...........................................................6 2.1. Implantation.............................................................................................. 6 2.1.1. Cytokines and implantation........................................................................ 6 Leukemia inhibitory factor .......................................................................... 6 Interleukin-1 ............................................................................................... 7 Macrophage-colony stimulating factor ....................................................... 8 HOXA10..................................................................................................... 8 2.1.2. Growth factors and implantation ................................................................ 9 Growth hormone ........................................................................................ 9 Epidermal growth factor ........................................................................... 10 Vascular endothelial growth factor ........................................................... 11 Insulin-like growth factor system .............................................................. 11 2.1.3. Blastocyst implantation and the adhesion cascade.................................. 13 Mucin-1 .................................................................................................... 13 Integrins ................................................................................................... 14 Selectins .................................................................................................. 16 Glycosylation-dependent cell adhesion molecule 1 ................................. 16 2.1.4. The role of uterine secretions during the pre-implantation period ............ 17 Uterine milk protein .................................................................................. 18 Osteopontin.............................................................................................. 19 2.1.5. Remodeling of the ECM at implantation................................................... 19 2.2. Maternal recognition of pregnancy and the pivotal role of IFN τττ........ 20 2.3. Mechanism of action of interferon tau on endometrial cells.............. 24 I Table of contents 2.4. Holistic approaches for the analysis of the transcriptome ................ 26 2.5. Global gene expression analyses of female reproductive tissues.... 30 2.6. The usage of monozygotic twin pairs as experimental animals........ 31 3. Materials and Methods .........................................................33 3.1. Materials ................................................................................................. 33 3.1.1. Chemicals ................................................................................................ 33 3.1.2. Enzymes and other reagents ................................................................... 33 3.1.3. Apparatuses............................................................................................. 35 3.1.4. Other Materials ........................................................................................ 36 3.1.5. Media and Solutions ................................................................................ 37 3.1.6. Oligonucleotides ...................................................................................... 39 3.1.7. DNA molecular weight markers................................................................ 40 3.2. Methods .................................................................................................. 41 3.2.1. Animals and tissue collection................................................................... 41 3.2.2. Standard methods used in molecular biology .......................................... 42 3.2.2.1. Isolation of total RNA from bovine endometrial cells................................ 42 3.2.2.2. Agarose gel electrophoresis..................................................................... 43 3.2.2.3. Precipitation of DNA................................................................................. 44 3.2.2.4. Phenol/chloroform extraction ................................................................... 45 3.2.3. Generation of a subtracted cDNA library.................................................. 45 3.2.3.1. Synthesis of cDNA for subtractive hybridization....................................... 45 First-strand synthesis............................................................................... 45 Second-strand synthesis.......................................................................... 46 3.2.3.2. Ligation of tester cDNA with the adapters................................................ 47 3.2.3.3. Subtractive Hybridization ......................................................................... 49 First step of subtractive hybridization....................................................... 49 Second step of subtractive hybridization.................................................. 49 Suppressor PCR ...................................................................................... 49 II Table of contents 3.2.3.4. Ligation with vector DNA.......................................................................... 50 cDNA preparation .................................................................................... 50 Vector preparation ................................................................................... 51 Ligation reaction and introduction into E. coli SURE© ............................. 52 Isolation of plasmid DNA.......................................................................... 52 3.2.4. cDNA library construction from subtracted cDNA produced by the vertis Biotechnologie AG ................................................................................... 53 3.2.5. Preparation of cDNA arrays ..................................................................... 53 Amplification of cDNA inserts via PCR..................................................... 53 Spotting the PCR products onto nylon membranes ................................. 54 3.2.6. Array hybridization with 33 P-labeled cDNA probes................................... 55 Synthesis of cDNA ................................................................................... 55 Radioactive labeling of the cDNA............................................................. 56 Prehybridization of arrays ........................................................................ 57 Hybridization ............................................................................................ 57 3.2.7. Analysis of array data .............................................................................. 58 3.2.8. Sequencing of cDNAs with differential hybridization signals and data analysis.................................................................................................... 58 3.2.9. Real-time RT-PCR ................................................................................... 59 3.2.10. Data Analysis of Real-time RT-PCR ........................................................ 61 3.2.11. In situ Hybridization.................................................................................. 61 4. Results...................................................................................63