Intercellular communication during in vitro maturation of bovine cumulus oocyte complexes [Elektronische Ressource] / Martin Fritz Schönfelder

Martin Schönfelder

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• Gesundheit

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2004
• Nombre de lectures: 28
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

Lehrstuhl für Physiologie
Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt
Technische Universität München



Intercellular communication during in vitro maturation
of bovine cumulus oocyte complexes



Martin Fritz Schönfelder


Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des
akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.


Vorsitzende: Univ.-Prof. Dr. Dora A. Roth-Maier


Prüfer der Dissertation: 1. Univ.-Prof. Dr. Ralf Einspanier,
(Freie Universität Berlin)
2. Univ.-Prof. Dr. Jürgen Polster


Die Dissertation wurde am 06.10.2003 bei der Technischen Universität München eingereicht und
durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und
Umwelt am 03.12.2003 angenommen.



"Neue Wege zeigen erfordert erst,
daß die alten gekannt werden.
Diese alten Wege wenigstens soweit zu kennen,
daß man in neue Gebiete eindringen kann, erfordert Zeit."

Rhoda Erdmann (1870-1935)























Für meine Frau
und meine Eltern


Table of contents

Chapter Page

ABBREVIATIONS I
1. ABSTRACT 1
2.ZUSAMMENFASSUNG2
3.GENERALINTRODUCTION 5
3.1.Oogenesis and follicle development 6
3.2. Maturation of oocyte 8
3.2.1. Oocyte maturation in vitro (IVM) 10
3.2.2. Spontaneous induction of oocyte 10
3.3. Factors influencing IVM success 11
3.3.1. Oocyte classification 11
3.3.2. Indicators of follicle and oocyte quality 12
4. LITERATURE REVIEW OF INVESTIGATED COMPONENTS 13
4.1. Steroidogenesis and endocrine disruption 13
4.2. Gap junctions 14
4.3. Nitric oxide 15
4.4. Growth factors 17
4.4.1. Growth differentiation factors-9 (GDF-9) and bone morphogenetic protein-15
(BMP-15) 17
4.4.2. Vascularendothelial growth factor (VEGF) 19
4.5. Extra cellular matrix components during oocyte maturation 20
5. AIMS OF THESTUDY23
6. MATERIALS AND METHODS 24
6.1. Tissue collection and preparation 24
6.1.1. COC retrieval and culture conditions 24
6.1.2. GC retrieval and culture conditions 24
6.1.3. Follicle dissection 25
6.2. Hormone determination 25

6.3. Reverse transcriptase polymerase chain reaction (RT-PCR) 25
6.3.1. Total RNA extraction 25
6.3.2. Reverse transcription of RNA 26
6.3.3. Primer design and PCR evaluation 26
6.3.4. Standard Block-PCR and gel electrophoresis 26
6.3.5. Quantitative real-time PCR 27
6.3.5. Intra- and inter-assay variation of LightCycler data 27
6.4. Cloning of growth and differentiation factor 9 28
6.4.1. Partial cDNA sequencing 28
6.4.2. Plasmid generation 28
6.4.3. Expression of recombinant protein 29
6.4.5. Isolation of recombinant natGDF9-protein by IMAC 29
6.4.6. SDS-poly acryl gel electrophoresis 30
6.4.7. Dialysis of protein solution 30
6.5. Histological examinations 31
6.5.1. Tissue preparation 31
6.5.2. Localization of hyaluronan
6.5.3. Immunolocalization of different antigens 31
6.5.4. Visual documentation of histological examinations 32
6.6. Buffers and primers 32
6.6.1 Modified Parker´s Medium 199 (MPM199) 32
6.6.2. Phosphate buffered saline (PBS) 33
6.6.3. List of used primers 33
6.6.4. Electrophoresis buffer for DNA gels 34
6.6.5. LB medium/agar (Luria-Bertani) 35
6.6.6. Buffers for protein isolation
6.6.7. 4x SDS sample buffer for SDS PAGE 35
6.6.8. 10x MOPS SDS PAGE running buffer 36
6.6.9. Coomassie staining solution 36
6.6.10. assie bleaching solution 36
7. RESULTS AND DISCUSSION 37
7.1. Steroidogenesis during IVM of bovine COC 37
7.2. Effects of TBT on steroidogenesis during IVM and GC culture 40
7.3. Presence of different connexins during IVM 42
7.4. Presence of NO synthases in bovine ovarian cells 45
7.5. GDF-9 and BMP-15 in maturing bovine oocytes 51
7.5.1. Expression of GDF-9 and BMP-15 in maturing COC 51
7.5.2. Expression of BMP receptors in maturing COC 52
7.5.3. Expression of BMP receptors in granulosa and theca cells 53
7.6. Sequencing the bovine GDF-9 cDNA, cloning and production of mature
recombinant GDF-9 protein 58
7.6.1. Sequencing of GDF-9 58

7.6.2. Production of recombinant mature GDF-9 59
7.6.3. Effects of recGDF-9 in GC culture 60
7.7.1. Effects of HA treatment on steroidogenesis and specific mRNA expression in
bovine granulosa cells 65
7.8. Vascular endothelial growth factor during IVM and embryo production 68
7.8.1. VEGF secretion of bovine GC under gonadotropins and hyaluronan 69
8. CONCLUSIONS 70
9. REFERENCELIST73
10. ACKNOWLEDGEMENTS90
APPENDIX91
App. 1. Internationally reviewed publications of the author 91
App. 1.1. Schoenfelder M, Schams D, Einspanier R.: Steroidogenesis during in vitro
maturation of bovine cumulus oocyte complexes and possible effects of
tri-butyltin on granulosa cells.
J Steroid Biochem Mol Biol. 2003 Feb; 84(2-3): 291-300. 91

App. 1.2. Schoenfelder M, Einspanier R: Expression of hyaluronan synthases and
corresponding hyaluronan receptors is differentially regulated during oocyte
maturation in cattle.
Biol Reprod. 2003 Jul;69(1): 269-77. 102

App. 1.3 Einspanier R, Schoenfelder M, Müller K, Stojkovic M, Kossmann M, Wolf E, and
Schams D: Expression of the vascular endothelial growth factor and its receptors
and effects of VEGF during in vitro maturation of bovine cumulus-oocyte
complexes (COC).
Mol Reprod Dev 2002, 62: 29-36. 112
App. 2. Papers in preparation of the author 121
App. 3. Abstract publications of the author 121
CURRICULUM VITAE 123



Abbreviations
Abbreviations

AC adenylate cyclase
ACC accession number of EMBL data base
ActR activin receptor
AG aminoguanidine hemisulfate salt
ALK activin receptor-like kinase
Appappendix
ARO aromatase P450
ATannealingtemperature
bFGF basic fibroblast growth factor
bHABP biotinylated hyaluronan binding protein
BMP-15 bone morphogenetic protein-15
BMPR bone morphogenetic protein receptor
BSA bovine serum albumin
cAMP cyclic adenosine mono phosphate
Cc cumulus cell
CDclusters ofdifferentiation
cDNA copy desoxyribonucleicacid
COC cumulus oocyte complex
Co-SMAD common-mediator SMAD
CP crossing point
Crcoronaradiata
Cx connexin
DDTdichloro-diphenyl-trichloroethane
DNA desoxyribonucleic acid
dNTPdesoxi nucleiotide triphosphate
DTT dithiothreitol
E2estradiol-17 β
eCG equine chorionic gonadotropin
ECM extra cellular matrix
EDeffectivedose
EDTA ethylenediamine tetra-acetic acid
EGF epidermal growth factor
EIA enzyme immuno assay
EMBL european molcular biology laboratories
eNOS endothelial nitric oxide synthase
ERK extra cellular-regulated-kinase
EtOHethanol
FA formaldehyde
FCS fetal calf serum
Fig. figure
I Abbreviations
flk fetal liver kinase
flt fms(feline sarcoma virus)-like tyrosin kinase
FSH follicle stimulating hormone
GAGglycosaminoglycan
GC granulosa cell
GD-VEGF glioma-derived vascular endothelial growth factor
GDF-9 growth and differentian factor-9
GMVB germinal vesicle breakdown
H1 histone 1
HAhyaluronan
HARE hyaluronan associated receptor for endocytosis
HAS hyaluronansynthase
hCG human chorion gonadotropin
HGF hepatocyte growth factor
HIShistidine
HRP horseradishperoxidase
HSD3-beta-hydroxy-steroid-dehydrogenase
ICAM intercellular adhesion molecule
ICSI intracytoplasmic sperm injection
IGF-1 insulin like growth factor-1
IHABP intracellular hyaluronan binding protein
IMAC immobilized metal affinity chromatography
iNOS inducible nitric oxide synthase
IPTG isopropyl-beta-D-thiogalactopyranoside
IVC in vitro culture
IVM in vitro maturation
kD kilo Dalton
KDRkinase domain region
KGF keratinocyte growth factor
KLkid ligand
lCc luteinizing cumulus cell
LH lteotropic hormone
L-NAMEN-omega-nitro-L-arginine methyl ester
LYVE lymph vessel endothelial hyaluronan receptor
MAD mother against dpp
MAPK mitogen activated protein kinases
MgCl2 magnesium chloride
MI/IImetaphaseI/II
MMLV Mouse Moloney murine leukemia virus
MOPS 3-[N-Morpholino] propanesulfonic acid
MPF maturation promoting factor
MPM modified Parker´s medium
II Abbreviations
mRNA messenger ribonucleic acid
nNOS neuronal nitric oxide synthase
NO nitric oxide
Oooocyte
P4 progesterone
PACEpairedbasic amino acid residue cleaving enzyme
PBS phosphate buffered saline
PCB polychlorinated bisphenyls
PCDDpolychlorinated dibenzodioxine
PCR polymerase chain reaction
PGE prostag