Investigations of extracellular matrix proteases, apoptotic and anti-apoptotic factors in the bovine corpus luteum [Elektronische Ressource] / von Heike Susanne Kliem

Heike Kliem

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 20
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Aus dem Lehrstuhl für Physiologie
der Technischen Universität München
(Betreuer: Prof. Dr. Dieter Schams)



vorgelegt über
Prof. Fred Sinowatz,
Lehrstuhl für Tieranatomie II
Ludwig-Maximillians-Universität München


Investigations of extracellular matrix proteases, apoptotic and anti-apoptotic
factors in the bovine corpus luteum


Inaugural-Dissertation

zur Erlangung der tiermedizinischen Doktorwürde
der Tierärztlichen Fakultät
der Ludwig-Maximilians-Universität München

von

Heike Susanne Kliem

aus

Friedberg / Hessen

München 2006

Gedruckt mit Genehmigung der Tierärztlichen Fakultät der
Ludwig-Maximillians-Universität München









Dekan: Univ.-Prof. Dr. E. P. Märtlbauer
1. Referent: Dr. Dr. F. Sinowatz
1. Korreferent: Univ.-Prof. Dr. H. Zerbe
2. Korreferent: Dr. W. Schmahl
3. Korreferent: Univ.-Prof. Dr. M. Förster
4. Korreferentin: Priv.-Doz. Dr. B. Schalch


Tag der Promotion: 28. Juli 2006



























Für
meine Eltern
Enrique
und
Frieda




















Contents
CONTENTS

LIST OF ABBREVIATIONS
1 INTRODUCTION AND AIM OF THE STUDY.............................................................12
2 LITERATURE .............................................................................................................14
2.1 REGULATION OF THE OESTROUS CYCLE IN CATTLE ................................................. 14
2.1.1 Folliculogenesis .......................................................................................... 14
2.1.2 Regulation of the oestrous cycle............................................................... 14
2.1.3 Corpus luteum (CL) formation, function and regression (luteolysis) .... 15
2.2 EARLY PREGNANCY IN CATTLE............................................................................... 16
2.3 ENDOCRINE REGULATORY FACTORS ...................................................................... 17
2.3.1 Progesterone ............................................................................................... 17
2.3.2 Oestradiol-17 β.............................................................................................. 17
2.3.3 Prostaglandin F2 α (PGF2 α) ........................................................................ 18
2.4 LOCALLY PRODUCED FACTORS .............................................................................. 18
2.4.1 Vascular endothelial growth factors (VEGFs) .......................................... 18
2.4.2 Housekeeping Genes.................................................................................. 19
2.4.3 Extracellular matrix proteases ................................................................... 20
2.4.3.1 General aspects...................................................................................... 20
2.4.3.2 Matrix metalloproteases (MMPs) ............................................................ 21
2.4.3.3 Tissue inhibitors of metalloproteases (TIMPs)........................................ 22
2.4.3.4 Plasminogen activator (PA) system........................................................ 23
2.4.3.5 Monocyte chemoattractant protein-1 (MCP-1) 23
2.4.4 Apoptosis..................................................................................................... 24
2.4.4.1 General aspects 24
2.4.4.2 Extrinsic pathway.................................................................................... 25
2.4.4.2.1 Tumor necrosis factor family (TNF α, TNFR1, TNFR2) ....................... 25
2.4.4.2.2 Fas and Fas-Ligand (FasL)................................................................. 27
2.4.4.3 Intrinsic pathway ..................................................................................... 28
2.4.4.3.1 Tumor suppressor p53........................................................................ 28
2.4.4.3.2 Bax and Bcl-X 29 L
2.4.4.3.3 Second mitochondria derived activator of caspases (Smac ) ............. 30
2.4.4.3.4 Survivin ............................................................................................... 30
2.4.4.4 Caspase3, -6, -7 31
3 MATERIAL..................................................................................................................32
3.1 DEVELOPMENT OF A METHOD TO GAIN UTERUS MILK IN VIVO ................................... 32
3.1.1 Equipment.................................................................................................... 32
3.1.2 Medicaments................................................................................................ 32
3.1.3 Biochemicals 33
3.1.4 Reagents ...................................................................................................... 33
3.2 EXPRESSION OF HOUSEKEEPING GENES, PROTEASES, APOPTOTIC AND ANTI-
APOPTOTIC FACTORS IN THE CL DURING OESTROUS CYCLE AND INDUCED LUTEOLYSIS
............................................................................................................................. 35
3.2.1 Tissue collection ......................................................................................... 35
3.2.1.1 Follicles 20 hours after GnRH application............................................... 35
3.2.1.2 CL during oestrous cycle ........................................................................ 35
3.2.1.3 CL during induced luteolysis................................................................... 36
3.2.2 Equipment 36
3.2.3 Surgical instruments................................................................................... 37
5 Contents
3.2.4 Medicaments................................................................................................ 37
3.2.5 Laboratory equipment................................................................................. 37
3.2.6 Biochemicals ............................................................................................... 39
3.2.7 Reagents ...................................................................................................... 40
3.2.8 Kits................................................................................................................ 40
3.2.9 Enzymes....................................................................................................... 40
3.2.10 Markers......................................................................................................... 41
3.2.11 Buffers and solutions.................................................................................. 41
3.2.12 Primersequences and polymerase chain reaction (PCR) products ....... 42
4 METHODS ..................................................................................................................46
4.1 DEVELOPMENT OF A METHOD TO GAIN UTERUS MILK IN VIVO ................................... 46
4.1.1 Enzyme immuno assay (EIA) of progesterone, oestradiol-17 β and PGF2 α
...................................................................................................................... 48
4.1.2 Radio immuno assay (RIA) of VEGF.......................................................... 50
4.2 EXPRESSION OF HOUSEKEEPING GENES, PROTEASES, APOPTOTIC AND ANTI-
APOPTOTIC FACTORS IN THE CL DURING OESTROUS CYCLE AND INDUCED LUTEOLYSIS
............................................................................................................................. 50
4.2.1 Transvaginal ovaryectomy ......................................................................... 50
4.2.2 Immunohistochemistry............................................................................... 50
4.2.2.1 Fixation and Paraffin embedding ............................................................ 50
4.2.3 Molecular techniques.................................................................................. 51
4.2.3.1 Enzyme immuno assay (EIA).................................................................. 51
4.2.3.2 RNA extraction........................................................................................ 51
4.2.3.3 DNA digestion......................................................................................... 52
4.2.3.4 Control of the RNA quantity and quality.................................................. 52
4.2.3.4.1 Quantity............................................................................................... 52
4.2.3.4.2 Quality................................................................................................. 53
4.2.3.5 Reverse transcription.............................................................................. 55
4.2.3.6 Polymerase chain reaction (PCR) methods............................................ 56
4.2.3.6.1 Block PCR........................................................................................... 56
4.2.3.6.2 Gradient PCR:........