Epimutations in Germ-Cell and Embryo Development [Elektronische Ressource] : Possible Consequences for Assisted Reproduction / Nady El Hajj. Betreuer: Thomas Haaf

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Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2011
• Nombre de lectures: 27
• Langue: Deutsch
• Poids de l'ouvrage: 10 Mo

Extrait

Epimutations in Germ-Cell and Embryo Development:
Possible Consequences for Assisted Reproduction









Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Bayerischen Julius-Maximilians-Universität Würzburg







vorgelegt von
Nady El Hajj




geboren in
Bkaatouta/Libanon


Würzburg Juli 2011






Eingereicht am:
Mitglieder der Promotionskommission:
Vorsitzender:
Gutachter: Univ.-Prof. Dr. med. Thomas Haaf
Gutachter: Univ.-Prof. Dr. med. Thomas Dandekar
Tag des Promotionskolloquiums:
Doktorurkunde ausgehändigt am:






























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Die vorliegende Arbeit wurde von November 2007 bis April 2011 an den Instituten für
Humangenetik der Universitäten Mainz und Würzburg unter Betreuung von Herrn
Univ.-Prof. Dr. med. Thomas Haaf angefertigt.

Hiermit erkläre ich, daß ich diese Doktorarbeit selbständig und ausschließlich unter
Verwendung der angegebenen Quellen und Hilfsmittel verfasst habe.

Die Dissertation hat bisher weder in gleicher noch in ähnlicher Form in einem
anderen Prüfungsverfahren vorgelegen.

Es wurde zuvor kein anderer akademischer Grad erworben.






Würzburg, Juli 2011 _______________________________

Nady El Hajj



























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Acknowledgements

First and foremost, I want to deeply thank my thesis advisor, Professor Dr. Thomas Haaf; for his
support and guidance. I appreciate all his contributions of time and ideas during my Ph.D. thesis.
I also would like to thank Professor Dr. Thomas Dandekar.

My sincere appreciation is extended to Dr.Ulrich Zechner and Dr. Eberhard Schneider for their
advice and encouragement and to all my colleagues at the Institute for Human Genetics at the
Universities of Mainz and Würzburg.

I wish to thank Dr. Rami Mahfouz and Dr. Nadine Darwiche who were great mentors during my
Masters studies.

I am really grateful to my uncle George Hage who has always been a great role model and source
of inspiration. Special thanks to my uncle Dr. Elias Karam and to Isabelle Karam for all their
help. Also, I would like to thank my good friends Amer, Jhonny, and Hicham.

Finally, I want to express my deepest gratitude to my parents, my brothers and sister, and
especially to my grandma for being my support system through the years. They offered never
ending love. To them I dedicate this thesis.



























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Outline

1. INTRODUCTION
1.1 Infertility: Causes and Effects .................................................................... 13
1.2 In-Vitro Ferilization: History and Usage ............................................................................ 15
1.3 ART and Imprinting Disorders ................................................................... 16
1.3.1 Large Offspring Syndrome..................................................................... 16
1.3.2 Angelman Syndrome ..................................................... .1.7. ....................
1.3.3 Beckwith-Wiedemann Syndrome ............................................................... 18
1.3.4 Prader-Willi Syndrome .........................................................................1..8.. .......................
1.3.5 Russel-Silver Syndrome ................................................9. ....................... 1
1.3.6 Retinoblastoma ............................................................... .2.0 ............
1.4 Genomic Imprinting .......................................................... .2.1 ................
1.4.1 Imprint Erasure ............................................................... .2.4. ...........
1.5 Repetitive Elements ............................................................................................. .2.8. ..............
1.6 Epigenetics and DNA Methylation ................................................................ 29
1.7 DNA Methylation and Infertility .................................................................. 31

2. MATERIALS AND METHODS
2.1 Materials ..................................................................................... 33
2.1.1 Sperm Samples for Quantitative Methylation Analysis, ........................ .3.3. .......................
2.1.2 Sperm Samples for Methylaion Array Analysis ..................................... .4.2 ............
2.1.3 Oocyte Samples .............................................................. .4.3. ............
2.1.4 Two Cell Embryo Samples .................................................................... 43
2.2 Methods ...................................................................................... 45
2.2.1 Sperm Purification......................................................... .4.5. .................
2.2.2 Sperm DNA Extraction ................................................4.5. .......................
2.2.3 Sperm DNA Quantification .............................................................................................. 46
2.2.4 Bisulfite Conversion of Sperm DNA ............................................................ 47
2.2.5 Cleanup of Bisulfite Treated Sperm DNA ................................................... .4.8. ..
2.2.6 Polymerase Chain Reaction (PCR) .............................................................. 49
2.2.7 Primer Design .................................................................. .5.4. .........
2.2.8 Gel Visualization ............................................................................................. .5.8. ..............
2.2.9 Pyrosequencing ................................................................................................................ 59
2.2.10 DNA Extraction and Bisulfite Conversion of Oocytes and Embryos ............................ 60
2.2.11 Limiting Dilution............................................................................................................ 62
2.2.12 Statistical Analysis ......................................................................................................... 63

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2.2.13 Protocol of Illumina Methylation Arrays ....................................................................... 64
2.2.14 Bioinformatics Analysis of Infinium Methylation Data ................................................ 70

3. Results
3.1 Methylation Analysis of Human Sperm DNA ............................................... .7.1. .....
3.1.1 Sperm Methylation of Paternally Imprinted Genes....................................... .7.1. ................
3.1.2 Sperm Methylation Analysis of Maternally Imprinted Genes ......................................... 72
3.1.3 Sperm Methylation Analysis of Repetitive Elements ............................ .7.2 ....................
3.1.4 Correlation of Aberrant Sperm DNA Methylation to Male Infertility ............................. 77
3.1.5 Boxplot Analysis of Sperm DNA Methylation ......................................... .7.9 ..........
3.1.6 Sperm DNA Methylation and ART Outcome ............................................ .8.0 ........
3.1.7 Predictive Power of ALU Methylation ........................................................... 81
3.1.8 Sperm DNA Methylation and Aging ............................................................ 83
3.2 Illumina Infinium Array Analysis of Sperm Methylation ........................4. ....................... 8
3.3 Methylation Analysis of Mouse Oocytes ............................................................................. 89
3.3.1 Oocyte DNA Methylation of the Pluripotency Marker Oct4 ........................................... 89
3.3.2 Oocyte DNA Methylation of Imprinted Genes ......................................... .8.9 ..........
3.3.3 Oocyte Lollipop Diagram...................................................................... 91
3.4 Methylation Analysis of Mouse Two-Cell Embryos ................................ .95. ................
3.4.1 Embryo DNA Methylation of the Pluripotency Marker Oct4.......................................... 95
3.4.2 Analysis of Maternally Imprinted Genes in 2-cell Embryos............................................ 95
3.4.3 Analysis of Paternally Imprinted Genes in 2-cell Embryos ............................................. 95
3.4.4 Limiting Dilution Efficiency in Single 2-cell Embryos ................................................... 96
3.4.5 Lollipop Diagram Representation of Embryo Methylation ............................................. 97

4. Discussion
4.1 The Effect of Sperm DNA Methylation on Infertility and F