A short-term suspension culture system for bovine oviduct epithelial cells suitable for the study of embryo-maternal communication processes [Elektronische Ressource] / by Regine Rottmayer

Regine Rottmayer

Description

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 Gedruckt mit Genehmigung der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Dekan: Univ.-Prof. Dr. E. P. Märtlbauer Referent: Univ.-Prof. Dr. E. Wolf Korreferent: Univ.-Prof. Dr. Dr. F. Sinowatz Tag der Promotion: 10. Februar 2006 Table of contents 1 Introduction...................................................................................................................... 1 2 Literature.......................................................................................................................... 2 2.1 Morphological characteristics of the bovine oviduct ............................................................ 2 2.1.1 Anatomy of the bovine oviduct....................................................................................................... 2 2.1.

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

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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

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Dekan:

Referent:

Korreferent:

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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

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Table of contents

1

2

3

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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  

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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

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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

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5.6

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Steroid responsiveness of cultured BOEC ........................................................................... 55

Conclusion...............................................................................................................................56

Summary ......................................................................................................................... 57

Zusammenfassung .......................................................................................................... 59

References ....................................................................................................................... 61

List of Figures ................................................................................................................. 74

List of tables .................................................................................................................... 75

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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 CO 2  CP

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.

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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