Regulation and functional signaling of decorin in the testis in health and disease [Elektronische Ressource] / Marion Adam. Betreuer: Lars Kunz

Marion Adam

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2011
• Nombre de lectures: 44
• Langue: Deutsch
• Poids de l'ouvrage: 18 Mo

Extrait

REGULATION AND FUNCTIONAL SIGNALING OF DECORIN
IN THE TESTIS IN HEALTH AND DISEASE


Marion Adam









Dissertation der Fakultät für Biologie
der Ludwig-Maximilians-Universität München


München 2011




Institut für Anatomie und Zellbiologie der Ludwig-Maximilians-Universität München





REGULATION AND FUNCTIONAL SIGNALING OF DECORIN
IN THE TESTIS IN HEALTH AND DISEASE








Marion Adam


















Diese Dissertation wurde angefertigt
unter der Leitung von Prof. Dr. Artur Mayerhofer
am Institut für Anatomie und Zellbiologie
an der Ludwig-Maximilians-Universität München





















Erstgutachter: PD Dr. Lars Kunz
Zweitgutachter: Prof. Dr. Michael Schleicher

Tag der Abgabe: 08.08.2011
Tag der mündlichen Prüfung: 15.11.2011




ERKLÄRUNG


Hiermit versichere ich ehrenwörtlich, dass meine Dissertation selbständig und ohne unerlaubte
Hilfsmittel angefertigt worden ist.
Die vorliegende Dissertation wurde weder ganz, noch teilweise bei einer anderen
Prüfungskommission vorgelegt.
Ich habe noch zu keinem früheren Zeitpunkt versucht eine Dissertation einzureichen oder an einer
Doktorprüfung teilzunehmen.


München, den 15.11.2011



i
TABLE OF CONTENTS

1. INTRODUCTION ...................................................................................................................................1
1.1 Male infertility...................................................................................................................................1
1.2 Testicular morphology......................................................................................................................2
1.3 Testicular cytoarchitecture and function .........................................................................................2
1.4 Regulation of testicular function......................................................................................................4
1.5 Peritubular myoid cells.....................................................................................................................6
1.6 Testicular fibrosis, mast cells and macrophages.............................................................................7
1.7 The proteoglycan decorin .................................................................................................................8
1.8 The cellular model ..........................................................................................................................12
1.9 The AROM+ mouse........................................................................................................................13
1.10 Aims of the study ..........................................................................................................................13
2. MATERIAL............................................................................................................................................15
2.1 Human biopsies...............................................................................................................................15
2.2 Transgenic mice expressing human P450 aromatase ...................................................................15
2.3 Rhesus monkey testicular samples.................................................................................................16
2.4 Other materials ................................................................................................................................16
3. METHODS .............................................................................................................................................17
3.1 Cell culture ......................................................................................................................................17
3.1.1 Isolation and cultivation of HTPC/-Fs................................................................................17
3.1.2 Isolation of mouse testicular fibroblasts .............................................................................17
3.1.3 NIH/3T3 cells .......................................................................................................................18
3.1.4 Cell treatment .......................................................................................................................19
3.2 Molecular biology...........................................................................................................................21
3.2.1 RNA-Isolation ......................................................................................................................21
3.2.2 Reverse transcription polymerase chain reaction...............................................................22
3.2.3 Semi-quantitative real-time PCR ........................................................................................24
3.2.4 Quantitative real-time PCR .................................................................................................25
3.2.5 Agarose gel electrophoresis.................................................................................................25
3.3 Protein biochemistry .......................................................................................................................26
3.3.1 Protein extraction and measurement of protein content ....................................................26
3.3.2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis ............................................26
3.3.3 Western blot..........................................................................................................................27
3.3.4 Enzyme-Linked Immunosorbent Assay..............................................................................28
3.3.5 Phosphorylation experiments: Proteome Profiler and Western blot.................................28 ii TABLE OF CONTENTS
3.4 Immunohistochemistry...................................................................................................................29
3.5 Immunocytochemistry....................................................................................................................30
3.6 Nuclear staining ..............................................................................................................................30
3.7 Measurement of cell viability and apoptosis.................................................................................31
3.7.1 ATP measurements ..............................................................................................................31
3.7.2 Caspase assay .......................................................................................................................31
3.7.3 CASY - measurements with an automated cell counting device......................................32
2+ 3.8 Measurements of intracellular Ca levels ....................................................................................32
3.9 Laser capture microdissection........................................................................................................33
3.10 Data analysis .................................................................................................................................33
4. RESULTS ...............................................................................................................................................34
4.1 DCN expression in the mammalian testis .....................................................................................34
4.1.1 Testicular DCN expression in the human, non-human primate and the mouse...............34
4.1.2 Testicular DCN expression in human sub-/infertility........................................................35
4.1.3 DCN expression in the testis of infertile AROM+ mice ...................................................36
4.1.4 Testicular DCN expression during rhesus monkey development.....................................38
4.2 Cultured human testicular peritubular cells – a cellular model to study male infertility...........39
4.2.1 Expression of smooth muscle cell- and fibroblast-markers ..............................................39
4.2.2 Expression of receptors in HTPCs and HTPC-Fs..............................................................41
4.3 Human testicular peritubular cells express DCN in vitro: Higher basal DCN levels in
HTPC-Fs than in HTPCs...............................................................................................................45
4.4 Regulation of DCN in human testicular peritubular cells......................................................