Gene expression of the testis-specific histone (H1t) in the spermatogenesis of the stallion [Elektronische Ressource] / submitted by Márcia Cristina Oliveira Cavalcanti

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• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2008
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

GENE EXPRESSION OF THE TESTIS-SPECIFIC
HISTONE (H1t) IN THE SPERMATOGENESIS
OF THE STALLION
MÁRCIA CRISTINA
OLIVEIRA CAVALCANTI
INAUGURAL DISSERTATIONédition scientifique
VVB LAUFERSWEILER VERLAG for the acquisition of the doctoral degree
at the Faculty of Veterinary medicine
VVB LAUFERSWEILER VERLAG ISBN 3-8359-5262-5
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From the Institute for Veterinary Anatomy -Embryology and -Histology
of the Justus-Liebig-University Giessen,

Supervisor: Prof. Dr. Martin Bergmann





Gene expression of the testis-specific histone (H1t)
in the spermatogenesis of the stallion


INAUGURAL DISSERTATION
for the acquisition of the doctoral degree
at the Faculty of Veterinary medicine
of the Justus-Liebig-University Giessen


submitted by
MÁRCIA CRISTINA OLIVEIRA CAVALCANTI
Veterinarian from Recife (Brazil)



Giessen 2008


oThe thesis was funded by the Graduate School N . 455
“Molecular Veterinary Medicine”, Germany Research Foundation (DFG)


With permission of the Faculty of Veterinary Medicine of the of the Justus-Liebig-University
Giessen



Dekan: Prof. Dr. Dr. Habil. G. Baljer
1. Referee: Prof. Dr. Bergmann
2. Referee: Prof. Dr. Schuler


Day of the Disputation: 04.February 2008











For my Family
(Deus é fiel)








„I declare that I have completed this dissertation single-handedly without the
unauthorized help of a second party and only with the assistence acknowledged therein. I have
appropriately acknowledged and referenced all text passages that are derived literally from or
are based on the content of published or unpublished work of others, and all information that
relates to verbal communications. I have abided by the principles of good scientific conduct
laid down in the charter of the Justus Liebig University of Giessen in carrying out the
investigations described in the dissertation.”








Márcia Cristina Oliveira Cavalcanti






1 LITERATURE SURVEY................................................................................................. 3
1.1 Anatomy and histology of the testis ..................................................................................3
1.1.1 Spermatogenesis…................................................................................................3
1.1.2 Mitosis and meiosis...............................................................................................6
1.1.3 Spermatocytogenesis.............................................................................................7
1.1.4 Spermiogenesis .....................................................................................................9
1.1.5 Sertoli cells .........................................................................................................10
1.1.6 Hormonal regulation ...........................................................................................10
1.1.7 Mammalian spermatogenic cycle, stage and wave ...............................................12
1.1.8 Spermatogenesis of the stallion (Equus caballus) ................................................12
1.1.9 Cryptorchism in the stallion ................................................................................17
1.2 Histone - Protamine replacement during spermatogenesis...............................................18
1.2.1 The linker histone ................................................................................................19
1.2.2 Transcriptional regulation of the histone H1t gene................................................20
1.2.3 Transcriptional activation of the H1t gene ............................................................21
1.2.4 Transcriptional repression of the H1t gene ...........................................................23
1.3 Aims of the study…………………………………………………………………………26
2 MATERIALS AND METHODS.....................................................................................26
2.1 General histological methods .........................................................................................27
2.1.1 Tissue collection ..................................................................................................27
2.1.2 Paraffin material ..................................................................................................27
2.1.3 Histology of the testicular samples………………………………………………..29
2.2 General molecular biology methods................................................................................34
2.2.1 RNA isolation with TRIzol reagent ...................................................................34
2.2.2 Reverse transcriptase polymerase chain reaction (RT-PCR) .................................36
2.2.3 Agarose gel electrophoresis…………39
2.2.4 Measurement of nucleic acid samples…………………………………………… 40
2.2.5 Real-Time RT-PCR………………………………………………………………. 41
2.2.6 Statistical analysis................................................................................................42
2.3 Partial cloning of the equine H1t mRNA ........................................................................43
2.4 In-situ hybridization of H1t in equine testis sections……………………………………..45
2.4.1 Production of digoxigenin (DIG)-labeled cRNA probes. .......................................45
2.4.2 In- situ hybridization……………………………………………………………….49
I 2.5 Antibody generation and verification ..............................................................................53
2.5.1 Histone isolation .................................................................................................54
2.5.2 Western blot analysis ..........................................................................................55
2.5.3 Immunohistochemistry……………………………………………………………63
2.7 General chemicals and reagents…………………………………………………………..67
2.7.1 Antibodies…………………………………………………………………………68
2.7.2 Equipment…………………………………………………………………………69
2.7.3 Other materials…………………………………………………………………….69
2.7.8 Kits…………………………….…………………………………………………..70
2.8 Abbreviations……….71
3 RESULTS ........................................................................................................................73
3.1 The testis specific histone (H1t) and ist expression in the testis………………………….73
3.1.1 H1t sequence and Genbank entries………………………………………………..73
3.1.2 Specific expression of the H1t mRNA .................................................................76
3.1.3 Quantitative H1t mRNA and statistical analysis ...................................................77
3.1.4 Cell-localization of H1t cRNAs in testis sections .................................................78
3.1.5 H1t protein expression and cell-protein-localization.............................................81
3.2 Age-dependent expression of the testis specific histone (H1t) .........................................85
3.2.1 Age-dependent H1t mRNA expression by RT-PCR.............................................85
3.2.2 Quantitative H1t mRNA expression and statistical analysis.................................86
3.2.3 Age-dependent H1t cRNA expression in testes sections ......................................87
3.2.4 Age-dependent H1t protein expression................................................................88
4 DISCUSSION ..............................................................