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DEVELOPMENTAL CHANGES IN GONADOTROPltf
RELEASING HORMONE NEURONS IN THE BRAIN OF THE
FEMALE RABBIT (oryctolagus cuniculus)
By
WARREN G. FOSTER, B.Sc., M.Sc.
A Thesis
Submitted to the School of Graduate Studies
in Partial Fulfilment of the Requirements
for the Degree
Doctor of Philosophy
McMaster University ,
(c) Copyright by Warren George Foster, November, 1990
DEVELOPMENTAL CHANGES IN THE GnRH NEURON
o
DOCTOR OF PHILOSOPHY (1990)Hamilton, Ontario
McMASTER UNIVERSITY(Health Sciences)
TITLE: Developmental Changes in Gonadotropin ReleasingHormone Neurons in the Brain of the Female Rabbit(Oryctolagus cuniculus)
AUTHOR: Warren George Foster, B.Sc. (Upiversity of Guelph)
M.Sc. (University of Guelph)
SUPERVISOR: Professor E.V. YoungLai
NUMBER OF PAGES: xv, 178
ii
ABSTRACT
This thesis examined developmental changes in morphology
of the GnRH neuron in the female rabbit brain during sexual
maturation. In the adult virgin rabbit approximately 1,000
GnRH cells were counted in half the hypothalamus. Two
morphologically distinct populations of GnRH neurons were
found. Fusiform cells with relatively smooth contours (smooth)
accounted for 34% of the total. Cells with irregular contours
(rough) represented 64% of the immunoreactive cells counted.
In a sUbsequent experiment GnRH cell types were quantified in
rabbits treated with Tamoxifen (TAM, lG ~g/kg/day), vehicle,
and pregnant mare serum (PHS, 50 IU on postnatal days (PND) 25
and 28). Sexual maturity was considered achieved when rabbits
attained a body weight of 3.0 kg. The proportion of rough
cells increased while the smooth cells decreased with sexual
maturation. This change was advanced by PHS treatment and
prevented by TAM treatment compared to controls. Sexual
maturity was advanced by PMS treatment (92 PND) versus controls
(108 PND) and delayed by TAM treatment (128 PND). Mean plasma
estradiol was significantly (P= 0.01) elevated in PHS rabbits
versus controls between PND 25 and 34 and again at PND 75 (p=
0.05). Since the total number of immunoreactive cells remained.- .-::~.
constant, it is concluded that smooth cells are transformed to
rough cells. In ano~her experiment chronic ovariectomy did not
change the total number of GnRH cells counted when compared to
.'
iv
sham operated rabbits. However, the developmental shift of
smooth cells to rough cells was prevented (p<O.05) in
ovariectomized rabbits. These results suggest that the
developmental change in GnRH cell morphology is functionally
related to puberty onset. Moreover, estradiol seems to induce
these changes through indirect mechanisms. It is proposed that
estradiol augments the growth neural imputs to GnRH cellls.
ACKNOWLEDGEMENTS
There are many people who were instrumental in making
my experience at McMaster enjoyable and successful as well
as contributing to the preparation of this thesis. First
and foremost I would like to thank my supervisor Dr. E. V.
YoungLai for his time and friendship over the preceding
four years. Dr. YoungLai has provided me with the
opportunity and freedom to pursue my thesis and non-thesis
research interests in a relatively independent manner,
which is most appreciated. Dr. YoungLai ' s support and
friendly discussions are gratefully acknowledged. Dr. J.
F. Jarrell, my cosupervisor is also deserving of many
thanks for his insights into the work described in this
thesis. I would also like to thank the other members of
my supervisory committee, Drs. G. Brown and A. Ball for
their time and help. I am especially indebted to Dr. Ball
for his instruction in immunohistochemical techniques and
critical evaluation of my work.
I would also like to pay special thank you's to Dr.
J. D. Booth, Roberta Petitti, Lisa Deys, Nicolle Thompson,
Cindy Todoroff, Joanne Gunby, Jan Yeo, Ann Schwidder,
Derick Waters, and Avril McMahon for the friendship that
has been extended to me whi Ie at McMaster. The many varied
discussions with Dr. Booth have been most enjoyable and
have served to broaden my perspectives and understanding
v
vi
of computers, endocrinology, and medicine. Jan Yeo's
technical skills of ovariectomy and radioimmunoassay are
greatly appreciated.
1 gratefully acknowledge the agencies ',Ilhich have
provided me with the following scholarships: ontario
Graduate Scholarship, Medical Research Council Studen~ship
and McMaster University Scholarship. The financial support
from scholarships has been very helpful in the pursuit of
my training.
Lastly, I thank my wife Helen for her support and
patience during my graduate study. For her understanding
Helen deserves more of a thank you than can be expressed
here.
TABLE OF CONTENTS
ACKNOWLEDGEMENTS <. • • v
LIST OF FIGtJR.ES "...... xi
LIST OF TABLES xiii
LIST OF ABBREVIATIONS ... .. ....... ...... ...... .. ...... .. xiv
CHAPTER IINTRODUCTION ................................................ 1
CHAPTER IINEUROENDOCRINOLOGY OF PUBERTY IN THE FEMALERABBIT: A REVIEW 9
Introduction .Puberty In The Rabbit .••••••••••..•••••••....•2.2.1 Physical Signs Of Puberty ••••••.•.•.••2.2.2 cirCUlating Gonadotropins ••..••••••.••2.2.3 Pituitary Responsiveness to Exogenous
2.2.8 Thg Gonadostat Theory .•••••••••.••.•••2.2.9 Synaptogenesis Theory •••••••••••••••••
Hypothalamic Localization Of GnRH ••....•••••.•2.3.1 Radioimmunoassay •••••••••.••••••••••.•2.3.2 Immunohistochemical ••••••••••••••.••••2.3.3 In situ Hybridization ••.•••••••••••••.
Regulation of GnRH Secretion •.•••.•••••••...••2.4.1 Catecholamines ••••••••••..•.••••••.•••
i) Morphological studies ••••••.•••••.ii) Functional Studies •.•••••.••••.•.•
2.4.2 Opioid Peptides •••••••.•.••••••••...••i) Morphological Studies ••••••••••.••ii) Functional Studies •••••••••••••.••
2.4.3 Gonadal steroids ••••••••••••.••.••••.•i) Morphological Studies ••.•.••••.•••ii) Functional Studies ••••••••••••••••
Summary •....•.•.....•.••..••••.••••••...•••...Hypothesis .Experimental Questions .
2.12.2
'.' 2.3
2.4
2.52.62.7
2.2.42.2.52.2 • ..32.2.7
GnRH .Ontogeny Of GnRH Receptors ..••••••••••Pulsatile Secretion Of Gonadotropins .•Pulsatile GnRH Secreti~n •••••••••••.••Ontogeny Of Ovarian Estrogens In theRabbit .
10111113
17212122
232430333335383939414346464749505153.57<;'57
vii
viii
CHAPTER IIIMATERIAl,S AND METHODS .••.••...•.•••••••.••......•.. 59
3.1 Animals & •••••••••••••••••• '" • • • 603.2 Experimental Design •••..••••••..•.•.••••.....• 60
3.2.1. Experiment I......................... 603.2.1.1 Localization Of Hypothalamic Nuclei. 603.2.1.2 Immunohistochemistry............... 62
3.2.1.2a Paraffin Technique •.•••••••.••.. 623.2.1.2b Thick sections •••.• -............ 653.2.1.2c Comparison Of Thick sections
Technique With Vibratome AndCryostat Methods ••••••••.••..••. 67
3 .2 .1. 3 Controls '" '" . . . . . . 693.2.1.4 Quantification Of Immunoreactive
GnRH. Neurons .••••.•.••••••.••••.•.•• 693.2.1.5 Topography Of GnRH Ueural Elements
In The Hypothalamus ..••••..••••.••••3.2.2. Experi~ent II '" .
3 • 2 • 2.1 Des~gn •••••••••••••••••••••••• '" •••••3.2.2.1a Evaluation Of Estradiol Effects On
Puberty And GnRH. Cytoarchitecture.3.2.2.1b Effect Of Ovariectomy On GnRH
cytoarchitecture •.••••••••••.•.•.3.2.2.2 GnRH Immunohistochemistry ••••••••••.3.2.2.3 Radioimmunoassays ••••.••••••••••••••3.2.2.4 Follicular Morphometry •••••••••.••.•3.2.2.5 Data Analysis ••.••••••••••••••••••••
3.2.3. Experiment III •••••••.•••••••••••..••••
CHAPTER IV
717171
71
737475777778
RESULTS ••••••••••••••••••••••••••••..••••.••••.••••• 79
4 • 1 EXPERIMENT I ..•••••••••.••.••••••.•••••••...•.• 804.1.1 Immunohistochemistry.................... 80
4.1.1.1 Paraffin Technique ••.••..••••••••••• 804.1.1.2 Thick sections •••••••••••••••••••••. 80
4.1.1.2a Specificity of Antisera ••••.••••• 804.1.2.2b Immunoreactive GnRH Cells •.•••••• 80
4,1.1.3 comparison of Thick vs. Vibratome andCryostat Techniques •••••••••••••••••• 81
4.1.1.4 Topography of Immunoreactive NeuralElements .•.••.•••.••••••••••.•.•... 86
4.2 EXPERIMENT II .•••••••••••••.••••••••••.•.•.•••• 894.2.1 Immunoreactive Cell Counts ••••••••••••• 894.2.2 Plasma and Ovarian Estradiol
Measurements ••••••••••••••••••••••.•••• 924.2.2.1 specificity of Estradiol
Antiserum............•.................. 92
ix
4.2.2.2 Developmental Chanes in Mean PlasmaEstradiol _. . . . . .. . . . . . . . 92
4.2.2.3 Developmental Changes in OvarianEstradiol 95
4.2.3 Developmental Changes in PlasmaGonadotropins •.••.••...••.•..••.•..••• 95
4.2.3.1 Plasma FSH ••••.•••.•••.....••..••.••.. 954.2.3.2 Plasma LH 984.2.3.3 FSH/LH Ratio •.•...•••.••....•••..••••. 1004.2.4 Physical Measurements .••.•..•...••...•. 1024.2.4.1 Body Weights.......................... 1024.2.4.2 Ovarian Weights ••••.•..••.••..•••.•••• 1024.2.4.3 Uterine and Pituitary Weights •••..••.• 1064.2.5 Mating Response •..•••.•••.•.•••.••••••. 1064.2.6 Follicle Morphometry •.•..••.••..•••..•• 1084.2.7 Ovariectomy Experiment •••••.•••••••.... 1104.2.7.1 Plasma Gonadotropins •..••..•...••••••. 1104.2.7.2 FSH/LH Ratio ..•..•••.•••..•.•••••••••• 1104.2.7.3 Immunoreactive Cell counts •••••..••••• 112
4.3 EXPERIMENT III •••••••••.••••••••.••••••.••••••• 112
CHAPTER VDISCUSSION 115
Proposed stages of Sexual Development in theRabbit .............•........................... 144Summary •.•••..••••..•••••...••••.••.•••.••••••• 148Conclusions 150
5.1 Introduction. .. . . .. .. . . .. . . . . . . . . .. . . . .. . . . . . . .. . . .. . . . 1165.2 Immunohistochemical Quantification, Morphological
Topography of GnRH Cells in the Brain of theAdult Rabbit .•.•.•..•...•...........•......•... 1185.2.1. specificity of GnRH Antisera .••••.••••• 1185.2.2. Quantification of GnRH Cells ••••.•••••. 119S~2.3. Topography of GnRH Neural Elements •.••• 124
5 •3 ..Developmental Changes in GnRH Neurons ••.••••••. 1275.3.1. Immunoreactive Cell Counts .•••••••••... 1275.3.2. Developmental Changes in Plasma Estradiol
and Gonadotropins ••••••••••.••••••••••• 1305.3.3. Relationship Between Body Weight and
Sexual Maturation in the Rabbit •••••••• 1355.3.4. Ovariectomy Impairs Developmental .
Changes in GnRH Neurons •••••••••••••••• 1395.3.5. Summary of Developmental Changes in the
GnRH Neuron 141Chronic Treatment with Tamoxifen Appears toPrevent the Development of Estrogen NegativeFeedback ..•••..••••••••••••••.••...•••..•.••.•• 144
5.4
-- 5.5
5.65.7
REFERENCES
x
.~ .
::,,~,
151
LIST OF FIGURES
Figure
Hypothalamic-pituitary-ovarian axis 3
2. Ontogeny of Serum Gonadotropins in theFemale Rabbit................................... 14
3. Amino Acid Sequence of GnRH •.•••.....•.........• 34
4. Models of Hypothalamic Neurocircuitry ...•...•... 56
5. Ventral View of Rabbit Hypothalamus •............ 64
6. Immunoreactive GnRH containing Cells of thePreoptic Area 82
7. GnRH cells clustered in the retrochiasmaticarea 82
8. MUltiple GnRH cell types seen in theretrochiasmatic area •.•...........•.••...•....•• 82
9. A smooth bipolar GnRH cell •.••••••..••.•.•.•••.• 83
10. and 11.process
Rough GnRH cells with mUltiple thickenedprotuberances . 83
12, 13a and 13b. Rough GnRH cells with spikedcontours 83
14. and 15. Camera lucida drawings of GnRH neuralelements in the rabbit hypothalamus (Fig. 14) •
. Hypothalamic nuclei (Fig. 15). 87
16. and 17. GnRH fibers in fr~ntal section •••.••••• 88
18. Immunoreactive fiber at ependymal surface •••.••• 88
19. Beaded swellings on GnRH-IR processes 88
20. to 23. Developmental changes in the number ofrough and smooth GnRH cells •••••••••..•••••••••. 91
24. Specificity of Estradiol Antisera ............... 93
25. Developmental changes in the mean plasmaestradiol levels .....••.•........•....••...•..•. 94
xi
xii
Figure
26. Developmental changes in ovarian estradiolcontent 96
27. Developmental changes in mean plasma FSH levels 97
28. Developmental changes in mean plasma LH levels 99
29. Graph of body weight changes during development 103
30. Cell counts in ovariectomized rabbits ••••••.••• 113
31. propos7d d7velopmental changes in hypothalamicneuroC1rcu1try 142
LIST OF TABLES
TABLE
I Summary of Reported Outcome Measures ofSexual Development in the Female Rabbit •.•.•.•.. 25
II Summary of Experimental Design •......••.••.••.. 61
III Comparison of Cell Counts Performed on Thickvs. Vibratome Sections ......•.•...•.••.••••.••. 85
IV Developmental Changes in FSH/LH Ratio ofTreated vs. Control Rabbits .•.•...••...•••••••. 101
V Mean Rate of Weight Gain .•...•.•...•••••••••.•.• 104
VI Developmental Changes in Body, Pituitary,Adrenal, uterine and Ovarian Weights ...•••••.•.•• 105
VII Mating Response .••.•••.•.•••.••...••.••••.•.•.• 107
VIII Developmental Changes in FollicularMaturation 109
"
IX Developmental Changes in the FSH/LH Ratio ofOvariectomized Rabbits Compared to Controls 111
X Effect of Tamoxifen Citrate on LH and FSHsecretion in the Adult Female Rabbit ••••.•••••• 114
xiii
ARCN
CNS
DA
DAB
E
EB
EOP
FSH
GABA
GH
GHRH
GnRH
GnRH-IR
h
hpg
IGF-I
i.v.
LH
MBH
ME
min
mRNA
mths
NAL
NE
LIST OF ABBREVIATIONS USED
arcuate nucleus
central nervous system
dopamine
diaminobenzidine
epinephrine
estradiol benzoate
endogenous opioid peptides
follicle stimulating hormone
gamma-amino-butyric acid
growth hormone
growth hormone releasing hormone
gonadotropin releasing hormone
gonadotropin releasing hormoneimmunoreactivity
hour
hypogonadal
insulin like growth factor I
intravenous
luteinizing hormone
medial basal hypothalamus
median eminence
minute
messenger ribonucleic acid
months
naloxone
norepinephrine
xiv