Characterization of voltage dependent anion channel (VDAC) subtypes in mammalian follicles and potential physiological relevance [Elektronische Ressource] / by Cassará, María Carolina

Sujets

• Gesundheit

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2007
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Characterization of Voltage Dependent Anion
Channel (VDAC) subtypes in mammalian
follicles and potential physiological relevance









Inaugural Dissertation
submitted to the
Faculty of Medicine
in partial fulfillment of the requirements
for the PhD-Degree
of the Faculties of Veterinary Medicine and Medicine
of the Justus Liebig University Giessen







by


Cassará, María Carolina

of

Buenos Aires, Argentina




Giessen 2007









From the Clinic of Dermatology and Andrology
Director: Prof. Dr. Peter Mayser
of the Faculty of Medicine of the Justus Liebig University Giessen






First Supervisor: Prof. Dr. Klaus-Dieter Hinsch
Second Supervisor: Prof. Dr. Aria Baniahmad










Committee Members: Prof. Dr. Heinz-Jürgen Thiel (Chairman)
Prof. Dr. Klaus-Dieter Hinsch
Priv.-Doz. Dr. Christine Wrenzycki
Prof. Dr.Andreas Meinhardt







thDate of Doctoral Defense: May 11 , 2007



















Para

Horacio, Ana, Paula y Gisella










List of abbreviations

List of abbreviations

°C degree Centigrade
µg microgram
µl icroliter
µm micrometer
AEC 3-amino-9-ethyl-carbazol
AS antiserum
AT annealing temperature
ATP adenosine triphosphate
2+Ca calcium ion
cDNA complementary DNA
cm centimeter
CO carbon dioxide2
COC cumulus-oocytes-complex
CP crossing point
DNA desoxyribosenucleic acid
et al. et alii
FITC fluorescein-5-isothiocyanat
GAPDH gyceraldehyde-3-phosphate dehydrogenase
gDNA genomic DNA
GV germinal vesicle
H2A Histone H2A
IVF In vitro Fertilisation
IVM In vitro Maturation
KCl potassium chloride
kDa kilodalton
M molar
mg milligram
MII etaphase II
min minute
ml milliliter
mm illimeter
mM millimolar

List of abbreviations

mRNA messenger RNA
MW molecular weight
NaCl sodium chloride
PAGE polyacrylamide gel electrophoreses
PBS phosphate Buffered Saline
PCR polymerase-chain-reaction
pH pondus hydrogenii
pI isoelectric point
PI preimmune serum
PMSF phenylmethylsulfonyl fluoride
RNA ribonucleic acid
RT-Reaction reverse transcription reaction
s second
SD standard deviation
SDS sodium dodecyl sulfate
TBS tris buffer saline
U2snRNA U2 small nuclear RNA
V volt
v/v volume per volume
VDAC voltage dependent anion channel
w/v weight per volume













Index
1 Introduction ....................................................................................................................... 8
1.1 Voltage Dependent Anion Channel (VDAC)................................................................. 8
1.1.1 VDAC structure and expression............................................................................. 9
1.1.2 VDAC localization............................................................................................... 10
1.1.3 Functional relevance of VDAC............................................................................ 10
1.1.4 VDAC in mammalian gametes 12
1.2 Fertilization................................................................................................................. 13
1.2.1 Spermatozoon....................................................................................................... 15
1.2.2 Oocyte .................................................................................................................. 16
1.3 Aims............................................................................................................................. 19
2 Material & Methods ........................................................................................................ 21
2.1 Expression of VDAC mRNA in porcine and bovine oocytes....................................... 21
2.1.1 Expression analyses of VDAC mRNA in porcine and bovine oocytes by RT-PCR
21
2.1.2 Real-time RT-PCR of bovine oocytes.................................................................. 24
2.2 Expression of VDAC protein isoforms in porcine gametes ........................................ 26
2.2.1 Total extraction of porcine spermatozoa proteins................................................ 26
2.2.2 Extraction of porcine oocyte protein.................................................................... 27
2.2.3 Generation of antibodies against subtype-specific synthetic VDAC peptides..... 28
2.2.4 Protein expression analysis of VDAC in porcine gametes by immunoblotting... 29
2.2.5 Protein identification by MALDI-TOF MS ......................................................... 32
2.2.6 Immunohistochemical detection of VDAC protein in porcine ovaries................ 32
2.2.7 Subcellular localization of VDAC proteins in porcine oocytes by confocal
microscopy......................................................................................................................... 33
2.3 Functional studies....................................................................................................... 34
2.3.1 Lipid bilayer experiments with purified VDAC protein from porcine spermatozoa
34
2.3.2 Treatment of mature porcine oocytes with purified VDAC protein from porcine
spermatozoa....................................................................................................................... 35
2.3.3 Calcium oscillation measurements after microinjection of purified VDAC protein
in mature bovine oocytes................................................................................................... 37
2.3.4 Influence of anti-VDAC1 antibodies on in vitro maturation of bovine oocytes.. 38
3 Results............................................................................................................................... 41
3.1 Expression of VDAC mRNA in porcine and bovine oocytes....................................... 41
3.1.1 Expression analyses of VDAC mRNA in porcine and bovine oocytes by RT-PCR
41
3.1.2 Real-time RT-PCR of bovine oocytes.................................................................. 42
3.2 Expression of VDAC protein isoform in porcine gametes.......................................... 47
3.2.1 Protein expression analyses of VDAC in porcine gametes by immunoblotting.. 48
3.2.2 Protein identification by MALDI-TOF MS ......................................................... 54
3.2.3 Immunohistochemical detection of VDAC protein in porcine ovaries................ 57
3.2.4 Subcellular localization of VDAC proteins in porcine oocytes by confocal
microscopy......................................................................................................................... 60
3.3 Functional studies....................................................................................................... 63


Index
3.3.1 Lipid bilayer experiments with purified VDAC protein from porcine
spermatozoa....................................................................................................................... 63
3.3.2 Treatment of mature porcine oocytes with purified VDAC protein from porcine
spermatozoa 65
3.3.3 Calcium oscillation measurements after microinjection of purified VDAC protein
in mature bovine oocytes................................................................................................... 67
3.3.4 Influence of anti-VDAC1 antibodies on in vitro maturation of bovine oocytes.. 68
4 Discussion ......................................................................................................................... 73
4.1 Expression of VDAC mRNA in porcine and bovine oocytes....................................... 73
4.1.1 Real-time RT-PCR of bovine oocytes.................................................................. 74
4.2 Expression of VDAC protein isoforms in porcine gametes ........................................ 76
4.2.1 Immununobiochemical detection and lipid bilayer experiments of VDAC proteins
from porcine spermatozoa ................................................................................................. 77
4.2.2 Immunobiochemical detection of VDAC proteins in porcine oocytes ................ 81
4.2.3 Immunohistochemical detection of VDAC protein in porcine ovaries 83
4.2.4 Subcellular distribution of VDAC proteins in porcine oocytes ........................... 84
4.3 Functional studies....................................................................................................... 87
4.3.1 Treatment of matured porcine