Expression of estrogen receptors alpha and beta, aromatase, steroid sulfatase and estrogen sulfotransferase in testes of immature and mature boars [Elektronische Ressource] / by Mutembei, Henry M
167 Pages
English
Downloading requires you to have access to the YouScribe library
Learn all about the services we offer

Expression of estrogen receptors alpha and beta, aromatase, steroid sulfatase and estrogen sulfotransferase in testes of immature and mature boars [Elektronische Ressource] / by Mutembei, Henry M'Ikiugu

Downloading requires you to have access to the YouScribe library
Learn all about the services we offer
167 Pages
English

Description

EXPRESSION OF ESTROGEN RECEPTORS ALPHA AND BETA, AROMATASE, STEROID SULFATASE AND ESTROGEN SULFOTRANSFERASE IN TESTES OF IMMATURE AND MATURE BOARSHENRY M'IKIUGU MUTEMBEIINAUGURAL DISSERTATIONsubmitted to the Faculty of Veterinary Medicine in partial édition scientifique fulfillment of the requirementsVVB LAUFERSWEILER VERLAGfor the PhD-Degree of the Faculties VVB LAUFERSWEILER VERLAG ISBN 3-8359-5079-7 of Veterinary Medicine and MedicineSTAUFENBERGRING 15D-35396 GIESSEN of the Justus Liebig University GiessenTel: 0641-5599888 Fax: -5599890 redaktion@doktorverlag.deFwww.doktorverlag.de 9 7 8 3 8 3 5 9 5 0 7 9 5édition scientifiqueVVB VVB LAUFERSWEILER VERLAGHENRY M'IKIUGU MUTEMBEI ESTROGENS AND THE BOAR TESTISDas 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 2006All 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.

Subjects

Informations

Published by
Published 01 January 2006
Reads 14
Language English
Document size 8 MB

Exrait

EXPRESSION OF ESTROGEN RECEPTORS ALPHA
AND BETA, AROMATASE, STEROID SULFATASE
AND ESTROGEN SULFOTRANSFERASE IN
TESTES OF IMMATURE AND MATURE BOARS
HENRY M'IKIUGU MUTEMBEI
INAUGURAL DISSERTATION
submitted to the Faculty of
Veterinary Medicine in partial
édition scientifique fulfillment of the requirements
VVB LAUFERSWEILER VERLAG
for the PhD-Degree of the Faculties
VVB LAUFERSWEILER VERLAG ISBN 3-8359-5079-7 of Veterinary Medicine and Medicine
STAUFENBERGRING 15
D-35396 GIESSEN of the Justus Liebig University Giessen
Tel: 0641-5599888 Fax: -5599890
redaktion@doktorverlag.de
Fwww.doktorverlag.de 9 7 8 3 8 3 5 9 5 0 7 9 5
édition scientifique
VVB VVB LAUFERSWEILER VERLAG
HENRY M'IKIUGU MUTEMBEI ESTROGENS AND THE BOAR TESTISDas 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 2006
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.
st
1 Edition 2006
© 2006 by VVB LAUFERSWEILER VERLAG, Giessen
Printed in Germany
VVB LAUFERSWEILER VERLAG
édition scientifique
STAUFENBERGRING 15, D-35396 GIESSEN
Tel: 0641-5599888 Fax: 0641-5599890
email: redaktion@doktorverlag.de
www.doktorverlag.de
Expression of Estrogen Receptors Alpha and Beta, Aromatase,
Steroid Sulfatase and Estrogen Sulfotransferase in Testes of
Immature and Mature Boars





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



by


Mutembei, Henry M’Ikiugu
of
Meru, Kenya



Giessen 2006From the of Clinic of Obstetrics, Gynaecology and Andrology for Small and Large
Animals with Ambulatory Service
Faculty of Veterinary Medicine, Justus-Liebig-University Giessen
Professur for Physiologie und Pathologie der Fortpflanzung II
Prof. Dr. Dr. hc Bernd Hoffmann








First Supervisor: Prof. Dr. Dr. h.c Bernd Hoffmann
Second Supervisor: Prof. Dr. Elvira Hinsch







Committee Members: Prof. Dr. Dr. h.c Bernd Hoffmann (Internal Thesis Examiner)
Committee Member: Prof. Dr. Dr. Ralf Einspanier (External Thesis Examiner)
Other committee Members: Prof. Dr. Dr. Hans Michael Piper (Chairman of Oral Panel),
Prof. Dr. Klaus Steger (Committee member) and Mr. Lambert Stamer (Secretary)





stDate of Doctoral Defense: 1 September 2006 DECLARATIONS






“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.”


Mutembei Henry M’Ikiugu
DEDICATIONS

TO MY WIFE MARY, AND MY KIDS, RON AND SHEILA

You all continuously showed me great love, patience, tolerance and understanding during
the entire period of our separation for this study. While each of these will be cherished
separately, they will be carried in my heart forever.
&
TO MY LATE BROTHER FRANCIS

Francis tirelessly stood by us since our daddy’s death in January 1984. He continuously
showed us kindness and compassion until his untimely demise in July 2005. May his soul
rest in peace and since history reminds us how little we know of those who came before us,
I will try to gather and preserve his legacy.
TABLE OF CONTENTS 1

TABLE OF CONTENTS
TABLE OF CONTENTS.................................................................................................... 1
LIST OF FIGURES............. 4
LIST OF TABLES............................................................................................................... 7
LIST OF ANNEXES........... 8
LIST OF IMPORTANT ABBREVIATIONS................................................................... 9
1. INTRODUCTION ................................... 11
2. LITERATURE REVIEW....................................................................................... 13
2.1 Anatomy and histology of the boar testis......................... 13
2.1.1 Location and gross morphology of the boar testis....................................... 13
2.1.2 Vascular supply and innervations ............................................................... 15
2.1.3 Temperature regulating system................................... 16
2.1.4 Brief histology of the boar testis.. 16
2.1.4.1 General histology .................................................................................................................... 16
2.1.4.2 Sertoli cells.............................. 17
2.1.4.3 Leydig cells 19
2.1.4.4 Spermatogonia......................... 20
2.1.4.5 Primary spermatocytes ............................................................................................................ 20
2.1.4.6 Secondary spermatocytes......... 20
2.1.4.7 Spermatids ............................................................... 21
2.1.4.8 Spermatozoon.......................................................................................... 21
2.1.4.9 Lamina propria........................ 22
2.2 Testicular and ductal system development and function............................... 22
2.2.1 Fetal period ................................................................................................................................. 22
2.2.2 Post natal and mature period...... 25
2.2.3 Spermatogenesis in the boar........ 26
2.2.3.1 Spermatogoniogenesis, stem cell renewal and spermatocytogenesis...... 27
2.2.3.2 Spermiogenesis........................................................................................................................ 28
2.2.3.3 Spermatogenic cycle, stage and wave...................... 29
2.2.3.4 Stages of spermatogenesis in the boar..................... 30
2.2.3.5 Sperm release (spermiation) and maturation.......................................................................... 36
2.2.3.6 Rate of sperm production in the boar...................... 36
2.3 Neuroendocrine regulation of spermatogenesis.............. 36
2.3.1 Sertoli-Germ cell interaction....................................................................................................... 36
2.3.2 LH, FSH, Testosterone................ 37
2.3.3 Estrogens ..................................... 37
2.4 Steroid hormones and their mechanisms of actions ....................................................................... 40
2.4.1 Overview on steroid hormones .................................... 40
2.4.2 Steroid hormone receptors and induction of transcription.......................... 42
2.4.3 Testicular steroid hormone synthesis................................ 45
2.5 Estrogen receptors (ERs) and their expression in the testis and epididymis................................ 48 2 TABLE OF CONTENTS

2.6 Functions of estrogen in the epididymis and testis ......................................................................... 50
2.7 Aims and objectives of the study ...................................... 52
2.7.1 Aim............................................... 52
2.7.2 Objectives.................................................................... 52
3. MATERIALS AND METHODS............................................................................ 53
3.1 Tissue Collection and Sample Preparation..................... 53
3.1.1 Experimental animals and design................................ 53
3.1.2 Sample collection and tissue preservation................................................... 53
3.2 Immunohistochemistry (IHC) .......................................................................... 54
3.2.1 Slide preparation......................... 54
3.2.2 Staining procedure...................................................... 54
3.2.3 Quantitative Evaluation of Positive Cells.................................................... 56
3.3 In vitro reverse transcription polymerase chain reaction (RT-PCR)............ 56
3.3.1 RNA isolation............................................................................................................................... 56
3.3.1.1 Extraction procedure............................................................................................................... 56
3.3.1.2 Measuring concentration......... 57
3.3.1.3 Standardisation of concentration and RNA storage................................ 57
3.3.2 RT Step of the in vitro RT-PCR.................................................................... 57
3.3.3 PCR step of the RT-PCR.............................................. 58
3.3.4 Analysis of the PCR-amplicons by Gel electrophoresis.............................. 61
3.4 One Step in situ RT-PCR for ER  and ER  ................................................... 62
3.4.1 Introduction and slide preparation................................ 62
3.4.2 In situ RT-PCR step ..................................................... 62
3.4.3 Washing and Immunostaining steps ............................................................ 63
3.5 In situ hybridization for StS and EST.............................................................. 63
3.5.1 Introduction ................................................................. 63
3.5.2 Generation of the probes ............................................................................. 63
3.5.3 In situ Hybridisation step............ 65
3.5.4 Washing and Immunostaining..... 66
3.6 Buffers and Solutions ........................................................................................................................ 67
3.7 Reagents and Sources 71
3.8 Materials, Equipments and their Sources ....................................................................................... 73
4. RESULTS................................................. 75
4.1 Expression of estrogen receptors...................................................................................................... 75
4.1.1 Immunolocalisation of ER by IHC............................... 75
4.1.1.1 Positive control experiments.... 75
4.1.1.2 Negative control for ER in porcine uterus............................................................................... 76
4.1.1.3 Negative control for ER in 50-day old boars........... 77
4.1.1.4 Negative control for ER in mature boars................. 77
4.1.1.5 ER  in 50 day old boars (immature)....................................................................................... 78
4.1.1.6 ER  in 50 day old boars.......................................... 81
4.1.1.7 ER  in sexually mature boars (250 day old group)................................ 82
4.1.1.8 ER  in the other groups (100-, 150- and 200-day old boars)................. 89
4.1.1.9 ER  in 100-day old boars....................................... 89
4.1.1.10 ER  in 150- and 200-day old boars.................................................... 90
4.1.1.11 ER  in sexually mature boars (250 day old group)............................. 92 TABLE OF CONTENTS 3

4.1.1.12 ER  in the other groups (100- 150- and 200- day old boars) ............................................. 97
4.1.1.13 ER  in 100- day old boars................................................................... 97
4.1.1.14 ER  in 150- and 200-day old boars..................... 98
4.1.2 Detection of ER-Specific mRNA................................ 100
4.1.2.1 In vitro RT-PCR................................................................................................ 100
4.1.2.2 ER localisation using in situ RT-PCR.................... 100
4.2 Expression of P450 aromatase by IHC.......................... 104
4.2.1 P450 aromatase in 50-day old boars......................................................................................... 104
4.2.2 P450 aromatase in 100-day old boars....................................................... 106
4.2.3 P450 aromatase in 150-day old boars 108
4.2.4 P450 aromatase in 200- and 250-day old boars ....................................................................... 110
4.2.5 Detection of aromatase-Specific mRNA.................... 111
4.3 Expression of Steroid sulfatase and estrogen sulfotransferase................................ 112
4.4.1 Immunolocalisation of Steroid Sulfatase (StS) by IHC.............................. 112
4.4.2 Detection of Steroid Sulfatase-Specific mRNA .......................................... 118
4.4.2.1 In vitro RT-PCR..................................................... 118
4.4.2.2 Porcine StS gene sequence and in situ hybridisation ............................................................ 118
4.4.3 Immunolocalisation of Estrogen sulfotransferase (EST) by IHC.............. 120
4.4.3.1 EST in 50-day old boars ........................................................................ 120
4.4.4 Detection of Estrogen sulfotransferase-specific mRNA (RT-PCR)............ 125
4.4.4.1 In vitro RT-PCR..................................................... 125
5. DISCUSSION......................................................................... 126
5.1 Experimental design........................................................ 126
5.2 Methods............................................................................................................ 126
5.2.1 Immunohistochemistry (IHC).................................... 126
5.2.2 In situ RT-PCR and in situ hybridisation................... 127
5.2.3 In vitro RT-PCR and sequencing............................................................... 128
5.3 Results............................................................................................................................................... 128
5.3.1 Estrogen Receptors.................... 128
5.3.2 Aromatase.. 133
5.3.3 StS and EST ............................................................................................................................... 133
6. THESIS ABSTRACT............................................................ 135
7. ZUSAMMENFASSUNG....................................................... 137
8. REFERENCES ...................................................................................................... 139
9. ANNEXES.............. 155
10. ACKNOWLEDGEMENTS .................................................................................. 158
11. CURRICULUM VITAE....................... 160

4 LIST OF FIGURES

LIST OF FIGURES
Fig. 1: Morphology of the boar testis ................................................................................................ 13
Fig. 2: Structure of Sertoli cell.......... 18
Fig. 3: Summary of genes essential for development of the testis .................................................... 23
Fig. 4: A Schematic representation of spermatogenesis .................................................................... 27
Fig. 5: Stages of spermatogenesis in the boar.................................................... 31
Fig. 5I: Stage 1 of spermatogenesis................................... 32
Fig. 5II: Stage 2 of spermatogenesis.................................................................. 32
Fig. 5III: Stage 3 of spermatogenesis ................................ 33
Fig. 5IV: Stage 4 of spermatogenesis 33
Fig. 5V: Stage 5 of spermatogenesis................................................................. 34
Fig. 5VI: Stage 6 of spermatogenesis ................................ 34
Fig. 5VII: Stage 7 of spermatogenesis............................... 35
Fig. 5VIII: Stage 8 of spermatogenesis ............................................................................................. 35
Fig. 6a: Schematic illustration of the structural and functional organisation of nuclear receptors ... 42
Fig. 6b: Structure of ER DNA binding domain ................................................................................. 43
Fig. 6c: Steroid hormone-receptor interaction................... 44
Fig. 7 Synthetic pathway of testicular steroid hormones................................................................... 45
Fig. 8: Comparison of the structure of ER  and ERβ proteins......................... 48
Fig. 9: pGEM®-T Vector and its multiple cloning sequences .......................................................... 64
Fig. 10a: ER  immunostaining in porcine uterus used as positive control....................................... 75
Fig. 10b: ER  immunostaining in porcine uterus used as positive control....... 76
Fig. 11a: Negative control slide in the porcine uterus....................................................................... 76
Fig. 11b: Negative control slide in the 50 day old boar testis........................... 77
Fig. 11c: Negative control slide in the mature boar testis ................................................................. 77
Fig. 12: Immunolocalisation of ER  in 50-day old boar testis......................... 78
Fig. 13: Immunolocalisation of ER  in 50-day old boar testis.......................................................... 81
Fig. 14a: ER  in 250-day boar testis during phase I of spermatogenesis ......................................... 83
Fig. 14b: ER  in 250-day boar testis during phase II of spermatogenesis........ 84