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From the Institute of Animal Breeding Veterinary Faculty of the Ludwig-Maximilian-University Munich Chair of Molecular Animal Breeding and Biotechnology Univ.-Prof. Dr. Eckhard Wolf
Supervisor: PD Dr. Stefan Hiendleder
A short-term suspension culture system for bovine
oviduct epithelial cells suitable for the study of
embryo-maternal communication processes
Inaugural Dissertation to achieve the Doctor Title of Veterinary Medicine at the Faculty of Veterinary Medicine of the Ludwig-Maximilian-University Munich
by Regine Rottmayer from Munich
Munich 2006
Dekan:
Referent:
Korreferent:
Gedruckt mit Genehmigung der Tierärztlichen Fakultät der
Ludwig-Maximilians-Universität München
Univ.-Prof. Dr. E. P. Märtlbauer
Univ.-Prof. Dr. E. Wolf
Univ.-Prof. Dr. Dr. F. Sinowatz
Tag der Promotion: 10. Februar 2006
Table of contents
1
2
3
Introduction ...................................................................................................................... 1
Literature .......................................................................................................................... 2
2.1 Morphological characteristics of the bovine oviduct ............................................................ 2
2.1.1 Anatomy of the bovine oviduct ....................................................................................................... 2
2.1.2 Epithelium of the bovine oviduct .................................................................................................... 3
2.2 Physiological functions of the bovine oviduct ........................................................................ 4
2.2.1 Gamete maturation, sperm capacitation and fertilization ................................................................ 4
2.2.2 Oviduct fluid ................................................................................................................................... 5
2.2.3 Transport mechanisms in the oviduct.............................................................................................. 6
2.3 Embryo-maternal communication.......................................................................................... 8
2.3.1
2.3.2
2.3.3
2.3.4
2.3.5
2.3.6
Embryo-maternal communication in the oviduct ............................................................................ 8
Growth hormone ............................................................................................................................. 9
IGF-system...................................................................................................................................... 9
Other growth factors...................................................................................................................... 10
Hyaluronic acid system ................................................................................................................. 11
Platelet-activating factor ............................................................................................................... 11
2.4 Somatic cells in culture .......................................................................................................... 12
2.4.1 Cell culture techniques .................................................................................................................. 12
2.4.2 Organ or organotypic culture ........................................................................................................ 13
2.4.3 Primary explant culture ................................................................................................................. 14
2.4.4 Cell culture .................................................................................................................................... 16
2.5 Bovine oviduct epithelial cells in culture .............................................................................. 17
2.5.1 Different cell preparation methods for BOEC............................................................................... 17
2.5.2 Co-culture experiments with embryos........................................................................................... 18
2.5.3 Studies on oviduct function........................................................................................................... 19
2.5.4 Quality assessment of BOEC in monolayer or suspension culture ............................................... 21
Material and methods .................................................................................................... 24
3.1 Equipment and expendable items ......................................................................................... 24
3.1.1 Cell preparation ............................................................................................................................. 24
3.1.2 Cell culture .................................................................................................................................... 25
3.1.3 Histological analyses..................................................................................................................... 25
3.1.4 Molecular analyses........................................................................................................................ 25
3.2 Used media and stock solutions............................................................................................. 26
3.2.1 Cell preparation ............................................................................................................................. 26
3.2.2 Cell culture .................................................................................................................................... 27
3.2.3 Electron microscopy...................................................................................................................... 28
3.2.4 RNA extraction ............................................................................................................................. 29
3.3 Animals.................................................................................................................................... 30
3.4 Culture of bovine oviduct epithelial cells obtained on Day 3.5 of the estrous cycle ......... 30
3.4.1 Oviduct preparation....................................................................................................................... 30
3.4.2 Preparation of cells........................................................................................................................ 31
3.4.3 Culture conditions and experiments .............................................................................................. 31
3.5 Morphological examinations ................................................................................................. 33
3.5.1 Trypan blue staining...................................................................................................................... 33
3.5.2 Immunocytochemistry................................................................................................................... 33
3.5.3 Light microscopy .......................................................................................................................... 34
3.5.4 Scanning electron microscopy....................................................................................................... 34
3.5.5 Transmission electron microscopy ................................................................................................ 35
3.6 Quantitative Real-time PCR examinations .......................................................................... 35
3.6.1 Total RNA extraction .................................................................................................................... 35
3.6.2 Reverse transcription quantitative PCR (RT-qPCR) ..................................................................... 36
3.7 Western blot analysis ............................................................................................................. 39
3.8 Statistical analysis .................................................................................................................. 40
4 Results ............................................................................................................................. 41
4.1 Cell yield, cell viability and purity of the epithelial cell culture......................................... 41
4.2 Cell morphology and ultrastructure..................................................................................... 42
4.3 RT-qPCR................................................................................................................................. 46
4.3.1 RNA isolation and RNA yield....................................................................................................... 46
4.3.2 Gene expression patterns during the course of culture.................................................................. 46
4.3.3 Influence of the supplementation with different serum................................................................. 48
4.3.4 Gene expression of cultured BOEC after stimulation with steroid hormones............................... 48
4.4
Western blot analysis ............................................................................................................. 50
5 Discussion ........................................................................................................................ 51
5.1 Stage of the estrous cycle ....................................................................................................... 51
5.2 Cells derived from oviducts ipsi- or contralateral to the ovulation site ............................ 51
5.3 Cell preparation and culture conditions .............................................................................. 52
5.4 Morphological findings .......................................................................................................... 53
5.5 Gene expression patterns during the culture period........................................................... 54
6
7
8
9
5.6
5.7
10
Steroid responsiveness of cultured BOEC ........................................................................... 55
Conclusion...............................................................................................................................56
Summary ......................................................................................................................... 57
Zusammenfassung .......................................................................................................... 59
References ....................................................................................................................... 61
List of Figures ................................................................................................................. 74
List of tables .................................................................................................................... 75
analysis of variance
annealing temperature
benzyldimethylamine
bovine oviduct epithelial cells
buffalo rat liver
bovine serum albumin
complementary DNA
carbon dioxide
AT
ANP
ANOVA
micrometer
microliter
degree Celsius
atrionatriuretic peptide
angiotensin II
DDSA
DMEM/F12
cDNA CO2CP
CS 3.5
BRL
BSA
BDMA
BOEC
ECS
E2
°C
Abbreviations
µm
µl
DNA
AngII
ET-1
FGF
ESR2
et al.
ESR1
e.g.
FITC
Fig.
g
GH
GPX4
G
crossing point
peroxidase
B. taurus non-selenium glutathione phospholipid hydroperoxide
growth hormone
gravity
gauge
fluorescein isothiocyanate
Figure
fibroblast growth factor
endothelin-1
and others
estrogen receptorβ
estrogen receptorα
estrous cow serum
estradiol-17β
for example
dodecenylsuccincanhydride
cow serum obtained on Day 3.5 of the estrous cycle
Dulbecco´s modified Eagle´s Medium / Hams F12
deoxyribonucleic acid
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