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