Qualitative and quantitative analysis of porcine sperm transcripts and characterization of a normalized cDNA library [Elektronische Ressource] / by Lucio Pereira Rauber

Lucio Rauber

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 20
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

From the
Department of Veterinary Sciences
Faculty of Veterinary Medicine
Ludwig-Maximilians-Universität München
Chair for Molecular Animal Breeding and Biotechnology
Prof. Dr. E. Wolf





Qualitative and quantitative analysis of porcine sperm transcripts
and characterization of a normalized cDNA library




Thesis for the attainment of the title Doctor in Veterinary Medicine
from the Faculty of Veterinary Medicine of the
Ludwig-Maximilians-Universität München



by
Lucio Pereira Rauber
from Santa Cruz do Sul - Brazil

Munich 2008


Gedruckt mit Genehmigung der Tierärztlichen Fakultät
der Ludwig-Maximilians-Universität München



Dekan: Univ.-Prof. Dr. Braun
Berichterstatter: Univ.-Prof. Dr. Wolf
Korreferent: Univ.-Prof. Dr. Heinritzi




Tag der Promotion: 18. Juli 2008
Aus dem
Department für Veterinärwissenschaften
Tierärztliche Fakultät
Ludwig-Maximilians-Universität München
Lehrstuhl für Molekulare Tierzucht und Biotechnologie
Prof. Dr. E. Wolf



Qualitative und quantitative Analyse porciner Spermientranskripte
und Charakterisierung der normalisierten cDNA-Bank




Inaugural Dissertation
zur Erlangung der tiermedizinischen Doktorwürde
der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München



von
Lucio Pereira Rauber
aus Santa Cruz do Sul – Brasilien

München 2008









Gedruckt mit Unterstützung des Deutschen Akademischen
Austauchdienstes
I Index
Index
Abbreviations.........................................................................................................................III
List of figures......................................................................................................................... V
List of tables ......................................................................................................................... VI
1. Introduction .............................................................................................................1
2. Literature.................................................................................................................3
2.1 Spermatogenesis ....................................................................................................3
2.1.1 Proliferative Phase ..........................................................................................3
2.1.2 Meiotic Phase..................................................................................................5
2.1.3 Differentiation ..................................................................................................5
2.1.4 Spermiogenesis...............................................................................................6
2.2 Transcripts, Proteins and their Regulation in Sperms..............................................6
2.2.1 Nucleus Proteins .............................................................................................6
2.2.2 Transcription ...................................................................................................7
2.2.3 Transcripts ......................................................................................................9
2.2.4 Posttranscriptional Regulation.......................................................................10
2.2.5 Alternative Transcripts...................................................................................10
2.2.6 Non-coding RNA ...........................................................................................11
2.2.7 Micro RNA and RNA Interference..................................................................12
2.2.8 PIWI-Interacting RNA ....................................................................................13
2.2.9 Translational Regulation................................................................................14
2.3 Chromatoid Body ..................................................................................................16
2.4 Residual Bodies....................................................................................................16
2.5 Ribosomal Proteins and Translation Factors.........................................................17
2.6 Apoptosis..............................................................................................................18
2.7 Cytoplasmic Bridges .............................................................................................19
2.8 Mitochondrion .......................................................................................................20
2.9 Polyspermy ...........................................................................................................21
2.10 Classical Techniques for Semen Evaluation..........................................................21
2.11 Molecular Biology..................................................................................................22
2.11.1 Overview.......................................................................................................22
2.11.2 Microarray techniques ...................................................................................25
2.12 Damaged DNA and paternal contribution ..............................................................25
3. Material and Methods............................................................................................27
3.1 Material.................................................................................................................27
II Index
3.2 Equipment.............................................................................................................27
3.2.1 Equipment for Gel Electrophoresis ................................................................27
3.2.2 PCR Equipment.............................................................................................28
3.2.3 Centrifuges....................................................................................................28
3.2.4 Chemicals .....................................................................................................28
3.2.5 Solutions and Reagents ................................................................................30
3.2.6 Water for PCR and RNA................................................................................32
3.2.7 Marker...........................................................................................................32
3.2.8 Reagents and Enzymes ................................................................................32
3.2.9 Other Materials..............................................................................................33
3.2.10 Oligonucleotides............................................................................................33
3.3 Methods................................................................................................................34
3.3.1 Agarose Gel Electrophoresis.........................................................................34
3.3.2 Sperm Recovery............................................................................................34
3.3.3 Handling RNA................................................................................................35
3.3.4 RNA Extraction..............................................................................................35
3.3.5 Generation of a Normalized cDNA Library.....................................................37
3.3.6 cDNA array Hybridization ..............................................................................41
4. Results..................................................................................................................44
4.1 RNA Extraction .....................................................................................................44
4.2 Normalized cDNA Library......................................................................................54
4.3 Comparative cDNA Array Hybridization of Sperm RNA from a Polyspermy Boar
and a Normal Control Boar....................................................................................62
5. Discussion ............................................................................................................67
5.1 Extraction and Characterization of RNA Isolated from Porcine Spermatozoa........67
5.2 Characterization of a Normalized cDNA Library Produced from Porcine
Spermatozoa.........................................................................................................70
5.3 Comparative Microarray Hybridization with Samples Derived from a Boar with
Polyspermy and a Control Boar.............................................................................75
5.4 Molecular Biology Tools for the Investigation of Infertility ......................................76
6. Summary ..............................................................................................................78
7. Zusammenfassung..........