Influence of peroxisomes on development, maturation and adult functions of the testis [Elektronische Ressource] / Anca Nenicu
178 Pages
English
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Influence of peroxisomes on development, maturation and adult functions of the testis [Elektronische Ressource] / Anca Nenicu

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178 Pages
English

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Influence of peroxisomes on development, maturation and adult functions of the testisANCA NENICUINAUGURAL DISSERTATIONsubmitted to the Faculty of Medicinein fulfillment of the requirementsfor the PhD-degree of the édition scientifique Faculties of Veterinary Medicine and MedicineVVB LAUFERSWEILER VERLAG of the Justus Liebig University GiessenVVB LAUFERSWEILER VERLAGSTAUFENBERGRING 15 ISBN: 978-3-8359-5617-9D-35396 GIESSENTel: 0641-5599888 Fax: -5599890redaktion@doktorverlag.dewww.doktorverlag.de 9 7 8 3 8 3 5 9 5 6 1 7 9édition scientifiqueVVB LAUFERSWEILER VERLAGVVB ANCA NENICU PEROXISOME IN TESTICULAR CELLSDas Werk ist in allen seinen Teilen urheberrechtlich geschützt. Jede Verwertung ist ohne schriftliche Zustimmung des Autors oder des Verlages unzulässig. Das gilt insbesondere für Vervielfältigungen, Übersetzungen, Mikroverfilmungen und die Einspeicherung in und Verarbeitung durch elektronische Systeme.1. Auflage 2010All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the Author or the Publishers.st1 Edition 2010© 2010 by VVB LAUFERSWEILER VERLAG, GiessenPrinted in Germany édition scientifiqueVVB LAUFERSWEILER VERLAGSTAUFENBERGRING 15, D-35396 GIESSENTel: 0641-5599888 Fax: 0641-5599890 email: redaktion@doktorverlag.

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Influence of peroxisomes on development,
maturation and adult functions of the testis
ANCA NENICU
INAUGURAL DISSERTATION
submitted to the Faculty of Medicine
in fulfillment of the requirements
for the PhD-degree of the
édition scientifique Faculties of Veterinary Medicine and Medicine
VVB LAUFERSWEILER VERLAG of the Justus Liebig University Giessen
VVB LAUFERSWEILER VERLAG
STAUFENBERGRING 15 ISBN: 978-3-8359-5617-9
D-35396 GIESSEN
Tel: 0641-5599888 Fax: -5599890
redaktion@doktorverlag.de
www.doktorverlag.de 9 7 8 3 8 3 5 9 5 6 1 7 9
édition scientifique
VVB LAUFERSWEILER VERLAGVVB
ANCA NENICU PEROXISOME IN TESTICULAR CELLSDas Werk ist in allen seinen Teilen urheberrechtlich geschützt.
Jede Verwertung ist ohne schriftliche Zustimmung des Autors
oder des Verlages unzulässig. Das gilt insbesondere für
Vervielfältigungen, Übersetzungen, Mikroverfilmungen
und die Einspeicherung in und Verarbeitung durch
elektronische Systeme.
1. Auflage 2010
All rights reserved. No part of this publication may be
reproduced, stored in a retrieval system, or transmitted,
in any form or by any means, electronic, mechanical,
photocopying, recording, or otherwise, without the prior
written permission of the Author or the Publishers.
st1 Edition 2010
© 2010 by VVB LAUFERSWEILER VERLAG, Giessen
Printed in Germany
édition scientifique
VVB LAUFERSWEILER VERLAG
STAUFENBERGRING 15, D-35396 GIESSEN
Tel: 0641-5599888 Fax: 0641-5599890
email: redaktion@doktorverlag.de
www.doktorverlag.de
Institute for Anatomy and Cell Biology II
Faculty of Medicine of the Justus Liebig University Giessen







Influence of peroxisomes on development,
maturation and adult functions of the testis







Inaugural Dissertation
submitted to the
Faculty of Medicine
in fulfillment of the requirements
for the PhD-degree
of the Faculties of Veterinary Medicine and Medicine
of the Justus Liebig University Giessen








Anca Nenicu


Giessen 2010

From the Institute for Anatomy and Cell Biology II
of the Faculty of Medicine of the Justus Liebig University of Giessen
Director / Chairperson: Prof. Dr. Eveline Baumgart-Vogt


















First Supervisor and Committee Member: Prof. Dr. Eveline Baumgart-Vogt

Second Supervisor and Committee Member: Priv.-Doz. Dr. Joachim Weitzel

Committee Members: Prof. Dr. Martin Diener

Priv.- Doz. Dr. Lutz Konrad













Date of Doctoral Defense: 20th August 2010
























My parents


























“If you doubt you can accomplish something, then you can't accomplish it. You have to have
confidence in your ability, and then be tough enough to follow through.”
Rosalyn Carter







Declaration


“I declare that I have completed this dissertation single-handedly without the unauthorized
help of a second party and only with the assistance 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.”


thGiessen, April 30 2010 Anca Nenicu


































List of Abbreviations


3 β-HSD 3 -hydroxysteroid dehydrogenase
17β-HSD 17 
17OH-P 17-hydroxypregnenolone
17OH-Pre 17-hydroxyprogesterone
ABC ATP-binding cassette family of transporters
ACOX Acyl-CoA oxidase
AMH Anti-Müllerian hormone
APS Ammonium persulfate
BSA Bovine serum albumin
CAT Catalase
cDNA Complementary deoxyribonucleic acid
COX Cyclooxygenase
CYP450arom Cytochrome P450 aromatase
CYP450scc Cytochrome P450 side-chain cleavage
°C Degree celcius
4-A androstenedione
5-A androstanediol
DHEA Dehydroepiandrosterone
DHT Dihydrotestosterone
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
DTT 1,4 ‐dithio‐DL ‐threitol
EDTA Ethylene‐diamine tetraacetate
ER Endoplasmic reticulum
FSH Follicle-stimulating hormone
GFP Green fluorescent protein
h Hour(s)
HSD Hydroxysteroid dehydrogenase
HTZ Heterozygote
IF Immunofluorescence
IHC Immunohistochemistry
IL Interleukin
KH PO Potassium dihydrogen phosphate 2 4
KO Knockout
LCFA Long-chain fatty acid
LH Luteinizing hormone
min Minute(s)
M Molar
MFP-2 Multifunctional protein-2
mg Milligram
ml Millilitre
Na HPO Disodium hydrogen phosphate 2 4
NaOH Sodium hydroxide
ng Nanograms
% Percentage
PBD Peroxisome biogenesis disorder
PBS Phosphate ‐buffered saline
PBST ‐buffered saline with Tween
PCR Polymerase chain reaction
Pex Gene encoding a peroxin (peroxisome biogenesis protein) PGE Prostaglandin
PFA Paraformaldehyde
PMP Peroxisomal membrane protein
PPAR Peroxisome proliferator activated receptors
PTS targeting signal
PUFA Polyunsaturated fatty acids
RNA Ribonucleic acid
ROS Reactive oxygen species
RT Room temperature
RXR Retinoic X receptor
SDS‐PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
s Second(s)
sER Smooth endoplasmic reticulum
SF-1 Steroidogenic factor 1
siRNA Small interfering RNA
SOD Superoxide dismutase
StAR Steroidogenic acute regulator protein
T Testosterone
TAE Tris acetate EDTA buffer
TEMED N, N, N, N ‐tetramethylethylenediamine
THIOLASE peroxisome 3-ketoacyl-CoA thiolase
Tris Tris (hydroxymethyl) aminomethane
μg Micrograms
μl Microliter
μm Micrometer
VLCFA Very long-chain fatty acid
v/v Volume/volume
WB Western blot
WT Wild-type
w/v Weight/volume
X-ALD X-linked Adrenoleukodystrophy
ZS Zellweger syndrome



Literature overview
1. Literature overview .......................................................................................................... 4
1.1. Overview on the male reproductive system ................................................................ 4
1.1.1. Structure of the adult testis.................................................................................. 4
1.1.2. Development of the testis.................................................................................... 4
1.1.3. The interstitial cells – Leydig cells ....................................................................... 5
1.1.3.1. Leydig cells - Target for hormones and mediator of hormone effects.............. 6
1.1.3.2. Growth factors – regulation of Leydig cells ..................................................... 6
1.1.3.3. Production of steroid hormones in Leydig cells............................................... 8
1.1.4. The testicular seminiferous tubule - structure and function................................ 11
1.1.4.1. The Peritubular myoid cells .......................................................................... 11
1.1.4.2. The Sertoli cell.............................................................................................. 11
I. Structure ................................................................................................................ 11
II. Maintenance of the integrity of the seminiferous epithelium ............................... 12
III. Functions of Sertoli cells ................................................................................... 13
1.1.5. Spermatogenesis .............................................................................................. 17
1.2. Peroxisomes............................................................................................................. 19
1.2.1. Nomenclature and morphology of peroxisomes ................................................ 19
1.2.2. Biogenesis of peroxisomes ............................................................................... 20
1.2.2.1. Peroxisomal matrix protein import and its receptors ..................................... 21
1.2.2.2. Lipid transport through the peroxisomal membrane...................................... 21
1.2.2.3. Peroxisomal functions .................................................................................. 22
1.2.2.4. Peroxisomal enzyme topology...................................................................... 22
1.2.2.5. Peroxisomes and its syndromes................................................................... 23
Deficiencies in peroxisome biogenesis .................................................................. 23
Peroxisomal single-enzyme deficiencies................................................................ 24
Peroxisomal dysfunction and male fertility ............................................................. 24
1.2.3. Mouse models for peroxisome dysfunction show impaired spermatogenesis.... 25
2. Materials and Methods .................................................................................................. 27
2.1. Human and animals tissue material used ................................................................. 27
2.1.1. Human .............................................................................................................. 27
2.1.2. Mice .................................................................................................................. 27
2.1.3. GFP-PTS1 transgenic mice............................................................................... 27
2.1.4. Necessary transgenic mouse lines for generation of Sertoli cell-specific Pex13
knockout mice (scsPex13KO) ..................................................................................... 27
• Pex13loxP – transgenic mice............................................................................ 27
• Amh-Cre – transgenic mice .............................................................................. 28
2.2. Breeding strategy of generation scsPex13KO mice using the Cre-loxP system........ 28
2.3. Genotyping with the polymerase chain reaction (PCR)............................................. 29
2.4. Laser micro-dissection of testes from 130 day-old mice scsPex13KO, scsPex13HTZ
and scsPex13WT ............................................................................................................ 30
2.5. Morphological experiments....................................................................................... 31
2.5.1. Fixation and embedding of the tissue................................................................ 31
2.5.2. Fixation and processing of testes for frozen sections ........................................ 32
2.5.3 Fixation and processing of tissue for electron microscopy – Cytochemical
localization of catalase activity with the alkaline DAB-method..................................... 32
2.5.4. Immunoelectron microscopy ............................................................................. 33
2.5.5. Immunohistochemistry (IHC)............................................................................. 33
2.5.6. Immunofluorescence (IF) .................................................................................. 34
2.5.7. Analysis of the specificity of catalase antiserum by antigen competition............ 34
2.5.8. Hematoxylin and eosin (H&E) staining .............................................................. 35
2.5.9. Oil Red O staining............................................................................................. 35
2.5.10 TUNEL assay................................................................................................... 35
2.6. Primary culture of somatic testicular cells................................................................. 36
2.6.1. Isolation and culture of Leydig cells................................................................... 36
2.6.2. Isolation and culture of peritubular myoid and Sertoli cells ................................ 37
1 / 169 Literature overview
2.7. Subcellular fractionation by differential centrifugation for the isolation of enriched
organelle fractions........................................................................................................... 38
2.7.1. Isolation of enriched peroxisomal fractions from primary cultures of Leydig-,
peritubular myoid- and Sertoli cells ............................................................................. 38
2.7.2. Isolation of enriched organelle fractions of interstitial, pertubular and tubular cells
of the testes of 130 day-old scsPex13KO, scsPex13HTZ and scsPex13WT mice ...... 39
2.8. Western blot analyses and relative quantification of protein bands........................... 41
2.9. RNA isolation and expression analysis by semi-quantitative RT-PCR ...................... 41
2.10. Blood collection ...................................................................................................... 42
2.11. Testis homogenates for steroids measurements .................................................... 42
2.12. Testis homogenate for very long chain fatty acid (VLCFA) and plasmalogen
measurements ................................................................................................................ 43
2.13. Fertility test for different scsPex13 mouse genotypes ............................................. 43
2.14. Pex13 silencing by RNA interference technology (RNAi) in primary Sertoli cell
cultures ........................................................................................................................... 43
2.15. ROS-detection by staining with dihydroethidium..................................................... 45
Secondary Antibodies ..................................................................................................... 47
3. Aims of the study........................................................................................................... 56
PART I. Peroxisomes in different cell types of testis in human and mice......................... 56
PART II. Physiological role of peroxisome in testis.......................................................... 56
4. Results............................................................................................................................ 58
4.1. Peroxisomal proteins are heterogeneously distributed in distinct cell types of the
mouse testis.................................................................................................................... 59
4.2. Cell type-specific differences in abundance of peroxisomal proteins are conserved
between mouse and man ................................................................................................ 61
4.3. Peroxisomes aggregate in clusters during spermatid maturation.............................. 62
4.4. The heterogeneity of peroxisomal enzymes is preserved in primary cell cultures and
cytospin preparations of isolated Leydig, peritubular myoid- and Sertoli cells.................. 67
4.5. Knockout of peroxisomal function in Sertoli cells ...................................................... 71
4.6. Fertility of scsPex13KO males.................................................................................. 73
4.7. Macroscopic differences between scsPex13WT, scsPex13HTZ and scsPex13KO
mice ................................................................................................................................ 73
4.8. Phenotypic differences of the testis and epididymis between scsPex13WT,
scsPex13HTZ and scsPex13KO mice at the microscopic level ....................................... 74
4.9. Analysis of semithin sections revealed pathological alterations in the testis of 130
day-old scsPex13KO animals.......................................................................................... 74
4.10. Electron microscopy confirms the severe pathological alteration in seminiferous
tubules and reveals ultrastructural changes also in Leydig cells...................................... 76
4.11. Specification of the accumulation of peroxisome - metabolized lipids in the testis of
scsPex13KO animals ...................................................................................................... 79
4.12. Impaired peroxisomal - and oxidation induced accumulation of fatty acids
primarily in Sertoli cells of scsPex13KO animals ............................................................. 80
4.13. Sertoli cells, spermatogenesis and the testicular integrity are progressively affected
during postnatal development of scsPex13KO animals ................................................... 83
4.14. Normal feature of prepubertal spermatogenesis in scsPex13KO............................ 83
4.15. Vacuolization of the cytoplasm of Sertoli cells in juvenile scsPex13KO .................. 84
4.16. Adult 60 day-old scsPex13KO mice exhibit hyperplasia of interstitial cells.............. 87
4.17. 90 day-old scsPex13KO mice display hypospermatogenesis ................................. 89
4.18. Pex13 gene deletion leads to “Sertoli cell only” syndrome in the testis of 130 day-old
mice ................................................................................................................................ 90
4.19. Immunofluorescence detection of steroidogenic enzymes in the testis ................... 96
4.20. The in vivo apoptosis rate of spermatogenic cells was strongly increased in 90 day-
old scsPex13KO mice ..................................................................................................... 98
4.21. Western Blots reveal the good quality of the tubular and interstitial cell preparation
...................................................................................................................................... 101
2 / 169
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