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This is to certify that the

dissertation entitled
The Gonadotropin Releasing HOrmone (GnRH) system
in Male Syrian Hamsters (Mesocricetus Auratus):
Organization and Regulation

presented by
Heather N. Ridhardson

has been accepted towards fulﬁllment
of the requirements for

Ph.D. degree inNenmscignge and Psychology

 

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major professor

Date 3/21/02

MS U is an Afﬁrmative Action/liq ual Opportunity Institution 0- 12771

 

 

 

LIBRARY

Michigan State

University

 

 

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THE GONADOTROPIN RELEASING HORMONE (GnRH) SYSTEM
IN MALE SYRIAN HAMSTERS (MESQQRICETUS AURATUS):
ORGANIZATION AND REGULATION
BY

Heather N. Richardson

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁlment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Psychology-Neuroscience Program

2002

ABSTRACT
THE GONADOTROPIN RELEASING HORMONE (GnRH) SYSTEM
IN MALE SYRIAN HAMSTERS (MESOCRICETUS AURATUS):
ORGANIZATION AND REGULATION
Heather NéRichardson

Neurons that synthesize and secrete the decapeptide
gonadotropin releasing hormone (GnRH) are the highest order of the
hypothalamic pituitary gonadal (HPG) axis, and thus, govern the entire
reproductive system. Many internal (e.g., steroid hormones) and
external (e.g., chemosensory cues) factors inﬂuence GnRH neuronal
activity, but mechanisms underlying regulation of these cells remain
elusive. GnRH neurons exist as anatomically distinct subpopulations of
cells in (rostral to caudal forebrain) tenia tecta, medial septum (MS),
diagonal band of Broca/organum vasculosum of the lamina terminalls
(DBB/OVLT), and caudal preoptic area (cPOA). The experiments of
this dissertation confirmed that a robust developmental event
(puberty), an internal hormonal stimulus (testosterone), and an
external sensory stimulus (female pheromones) in male Syrian
hamsters all affect GnRH neurons in a brain region-dependent manner.

The most rostral cell groups were robustly affected by puberty

and testosterone. These same cells groups also had a higher

proportion of close appositions from GnRH ﬁbers, suggesting more

abundant GnRH-GnRH communication within these cell groups. The
most caudal cells (cPOA) may be important for generating the
neuroendocrine response to female chemosensory cues, as this is the
brain area that has the highest proportion of close appositions from
ﬁbers of the medial amygdala, a chemoensory processing nucleus.

In summary, there is heterogeneity within the GnRH system.
GnRI-l cell populations respond to puberty and to internal and external
stimuli differently depending on where they reside in the brain.
Communication within the GnRH system and between non-GnRH and
GnRH neurons also varies with brain region. Future work determining
the types of connections within the GnRH system (synaptic or non-
synaptic), the phenotype of neuronal or non-neuronal cells projecting
to the GnRH system, and changes in receptor expression within these
different GnRH subpopulations would advance understanding of how

these variables are integrated by GnRH system to impact reproduction.

This dissertation is dedicated to my parents

and to Samuel, for all of your love and support

ACKNOWLEDGMENTS

It has been a privilege to work with the staff, students, and
faculty at Michigan State University. I especially thank my graduate
advisor and mentor, Dr. Cheryl Sisk. I thank you for challenging me
and inspiring me to succeed. Your professional advice, guidance, and
support have been invaluable to my career in science. Many thanks to
my committee members, Drs. Laura Smale, Juli Wade, and Jim
Galligan for your detailed comments on my research and writing. I
greatly appreciate all of the advice you have provided throughout my
tenure at Michigan State University.

This dissertation is the result of a great deal of work on the part
of many people over the years. I would like to thank my colleague and
good friend, Dr. Russell Romeo, for all of his help and support during
my graduate career. Thanks also to Jane Venier, Dr. Dave Parfitt, Dr.
Eman Ahmed, Dr. Yu Ping Tang, Kalynn Schulz Wilson, Kallris Salas-
Ramirez, Aaron Nelson, and Kristina Davis for all of your help along the
way. This dissertation would have not been possible without you.
Thanks to Drs. Heather Eisthen, Keith Lookingland, and Cindy Jordan

for the use of their lab space, equipment, and expertise.

TABLE OF CONTENTS

LIST OF FIGURES ................................... viii
LIST OF TABLES ................................... xiv
CHAPTER 1:

GENERAL INTRODUCTION ......................... 1
CHAPTER 2:

DISTRIBUTION OF GnRH NEURONS AND PUBERTAL

CHANGES IN IMMUNOREACTIVITY .................. 13

Methods ..................................... 15

Results ...................................... 24

Discussion ................................... 25
CHAPTER 3:

DOES TESTOSTERONE REGULATE GnRH mRNA? ......... 31

Methods ..................................... 36

Results ...................................... 44

Discussion ................................... 54
CHAPTER 4:

THE INFLUENCE OF TESTOSTERONE ON GNRH mRNA

BEFORE AND AFTER PUBERTY ...................... 60

Methods ..................................... 63

Results ...................................... 70

Discussion ................................... 78
CHAPTER 5:

PHEROMONES AND THE GnRH SYSTEM ............... 83

Methods ..................................... 86

Results ...................................... 95

Discussion ................................... 102
CHAPTER 6:

ANATOMICAL RELATIONSHIPS AMONG GNRH NEURONS

AND MEDIAL AMYGDALA EFFERENTS ................. 108

Introduction .................................. 108

Methods ..................................... 111

vi

Results ...................................... 122

Discussion ................................... 135
CHAPTER 7:

GENERAL DISCUSSION .......................... 140
REFERENCES ...................................... 151

vii

LIST OF FIGURES

Figure 1. Diagram of the hypothalamic-pituitary gonadal (HPG) axis.

Figure 2. Camera lucida line drawings depicting the three regions
analyzed: DBB/OVLT, MS, and cPOA (A, dashed boxes). Asterisks
represent the location of GnRH+ cells in a representative animal.
Abbreviations: CC, corpus callosum; LV, lateral ventricle; AC, anterior
commissure; II, optic nerve; 3V, 3rd ventricle; OVLT, organum
vasculosum of the lamina terrninalis; OC, optic chiasm.
Photomicrographs depicting immunocytochemical staining of
ambiguous cells (B, arrows) and GnRH cells (C, arrows) using LR-1
(1:10,000). Bar, 100 um.

Figure 3. mean (:l:SEM) number of GnRH+ cells/section in the MS
(A), DBB/OVLT (B), and cPOA (C) of juvenile and adult males in which
brain tissue was stained with a low (1:40,000) or high (1:10,000)
dilution of LR-1 antiserum. Letters in panel A indicate a signiﬁcant
main effect of age, with juvenile males (“a") having signiﬁcantly more
GnRH+ cells/section in the DBB/OVLT than adult males (“b”)
independent of antiserum dilution (ps0.05). Letters in panel B indicate
that juvenile males (“a") had signiﬁcantly more GnRH+ cells/section
than adult males (“b”) only with the 1:40,000 dilution of LR-1

(ps0.0S).

Figure 4. A: Drawing of an anatomical section (adapted from Morin
and Wood, 2001) delineating the location of GnRH cells in the tenia
tecta. B: GnRH-immunoreactive cells and ﬁbers in the tenia tecta
(see Chapter 2 for details on GnRH immunocytochemistry methods).
C: GnRH-mRNA labeled cells in the tenia tecta. Differences in section
thickness (40 pm for immunocytochemistry vs. 10 pm for ISSH) likely
account for the different number of cells in the two

photomicrographs. Bar, 20 um.

Figure 5. Drawing of a sagittal view of the male Syrian hamster brain
summarizing four GnRH subpopulations: tenia tecta (Tl'), medial
septum (MS) diagonal band of Broca/organum vasculosum of the
lamina terrninalis (DBB/OVLT), and caudal preoptic area (cPOA). Red
boxes delineate the boundaries of each subpopulation.

viii

Figure 6. Camera lucida drawings of the most anterior and posterior
sections of the four brain regions analyzed: tenia tecta, medial septum
(MS), diagonal band of Broca/organum vasculosum of the lamina
terminalis (DBB/OVLT), and caudal preoptic area (cPOA). The area
within which the GnRH cells reside is indicated by shading.
Abbreviations: II, optic nerve; 3V, 3rd ventricle; AC, anterior
commissure; CC, corpus callosum; LV, lateral ventricle; 0C, optic
chiasm.

Figure 7. Plasma testosterone (top panel) and LH (bottom panel)
levels in juvenile and adult males following castration and treatment
with O or 2.5 mg testosterone. Asterisk and bars indicate a main
effect of testosterone treatment on plasma testosterone and LH
(ps0.05) and no interaction within age. Thus, hamsters treated with
2.5 mg testosterone had signiﬁcantly higher plasma testosterone than
hamsters treated with 0 mg testosterone (top panel). Hamsters
treated with 2.5 mg testosterone had signiﬁcantly lower plasma LH
than hamsters treated with 0 mg testosterone (bottom panel).

Figure 8. Photomicrographs of labeled cells in the tenia tecta from
castrated adults treated with 0 mg testosterone (A) or 2.5 mg
testosterone (B). Only silver grains over the Nissl stain were
measured (dark grains in plane of focus), providing a conservative
estimate of the total silver grain staining associated with each cell.
Bar, 20 um.

Figure 9. Mean grain area (umz) in the tenia tecta, DBB/OVLT, MS,
and cPOA in castrates (collapsed across age) treated with 0 or 2.5 mg
of testosterone. Asterisk indicates a signiﬁcant effect of treatment
only in the tenia tecta (ps 0.05). Testosterone treatment signiﬁcantly
reduced GnRH mRNA mean grain area within this group of GnRH
neurons.

Figure 10. Frequency distribution of the percentage of GnRH neurons
within the tenia tecta, DBB/DVLT, MS, and cPOA in each category of
GnRH mRNA grain area (umz). Mean grain area for each analyzed cell
was rounded to the nearest whole number and the cells were
categorized into bins ranging from 1 pm2-36 umz. The total number of
cells for each treatment group determined n. Data from both juveniles
and adults were included in the treatment groups. Frequency

ix

distributions for castrates and testosterone-treated males were
signiﬁcantly different (Ps.001) only in the tenia tecta and MS.

Figure 11. Simple regression analyses of GnRH mRNA mean grain
.area (umz) and plasma LH for the tenia tecta, DBB/OVLT, MS, and
cPOA. All age and treatment groups are represented in these analyses
and each dot represents data from an individual animal. There was a
signiﬁcant correlation (Ps.05) between plasma LH and GnRH mRNA
mean grain area only within the tenia tecta.

Figure 12. Camera lucida drawings of the tissue dissection procedure.
Asterisks mark the three tissue blocks collected for the RNAse
protection assay: tenia tecta and medial septum (TT/MS), DBB/DVLT,
and cPOA. Abbreviations: II, optic nerve; 3V, 3rd ventricle; AC,
anterior commissure; CC, corpus callosum; LV, lateral ventricle; 0C,
optic chiasm; ME, median eminence.

Figure 13. GnRH mRNA RNAse protection assay reference standards
(A) and standard curve (B).

Figure 14. Plasma testosterone levels following testosterone
treatment. Treatment signiﬁcantly increased plasma testosterone in a
dose dependent manner (ps 0.05). Juveniles and adults did not differ in
plasma testosterone levels with any close.

Figure 15. Plasma LH levels following treatment with different doses
of testosterone. Plasma LH was signiﬁcantly reduced by testosterone
treatment in a dose dependent manner (ps0.0S). Adults had
signiﬁcantly higher plasma LH than juveniles (p50.05).

Figure 16. Representative examples of GnRH mRNA levels in the
TT/MS, DBB/OVLT, and cPOA of gonadectomized juvenile and adult
males exposed to 0, 0.5, 1.5, or 2.5 mg testosterone pellets.

Figure 17. Mean GnRH mRNA levels in the Tr/MS, DBB/OVLT, and
cPOA following treatment with testosterone. Age and treatment
effects were signiﬁcant only in the ‘lT/MS (ps0.05). Within the Tr/MS
adults had higher mean levels of GnRH mRNA and testosterone
treatment reduced mean levels of GnRH mRNA in a dose dependent
manner.

Figure 18. Representative example plasma LH proﬁles following
exposure to a cotton swab containing pheromone (A) or a control swab
(B). The arrows indicate delivery of the swab (pheromone=black
arrow, blank=clear arrow).

Figure 19. Time response curves of mean (:l: SEM) plasma LH (%
change from baseline) following exposure to pheromone or blank
swabs. There was a signiﬁcant increase in plasma LH only after
exposure to pheromone (ps0.05).

Figure 20. Mean (:l: SEM) plasma LH levels (ng/ml) 0, 60, and 120
min following exposure to swabs containing pheromone. There was
not a signiﬁcant effect of pheromone on LH across time (p>0.05).

Figure 21. Mean (:l: SEM) plasma testosterone levels (ng/ml) 0, 60,
and 120 min following exposure to swabs containing pheromone.
Testosterone was signiﬁcantly elevated 60 min after exposure

(ps 0.05).

Figure 22. Mean area of silver grains (umz) as measured within the
boundary of the cell body (left graphs) and within and surrounding the
cell body (right graphs) in the tenia tecta, MS, DBB/OVLT, and cPOA of
pheromone exposed males. Pheromones did not affect GnRH mRNA,
as measured by either index, at any time point following exposure

(p>0.05).

Figure 23. Photomicrographs showing medial amygdala BDA injection
sites in representative animals killed 2, 4, 7, and 14 days after
surgery. Survival times of 2 and 4 days resulted in diffuse spread of
BDA. The injection sites were more distinct 7 and 14 days after
surgery.

Figure 24. Schematic representations of animals with BDA injections
into the medial amygdala (A,#806; B, #809) and substantia
innominata (C, #810). Animal #806, #812, and #810 had BDA-GnRH
ratios of 1.0, 0.9, and 0, respectively, in the cPOA ipsilateral to the
injection. Abbreviations: ACo, anterior cortical nucleus of the
amygdala; AHA, amygdalohippocampal area; BM, basolateral nucleus
of the amygdala; Ce, central nucleus of the amygdala; IM,
intercalcalated mass cell; MeA, anterior medial

xi

amygdala; MeP, posterior medial amygdala group; oc, optic chiasm;
ot, optic tract; PMCo, posteromedial cortical nucleus of the
amygdala; PLCo, posterolateral cortical nucleus of the amygdala,

si; substantia innominata.

Figure 25. GnRH and BDA ﬁbers in the stria terminalis. Bar, 20 um.

Figure 26. GnRH (gray arrows) and BDA (black arrows) double labeled
ﬁber. Bar, 20 um.

Figure 27. Photomicrographs of a GnRH cell in the cPOA viewed in
different focal planes. The black arrow indicates a BDA close
apposition and gray arrows indicate GnRH close appositions onto the
same cell. Bar, 10 um.

Figure 28. Photomicrograph of a GnRH neuron in the tenia tecta with
a BDA apposition onto the axon. Also note the second GnRH cell body
in close apposition to this GnRH neuron. Bar, 10 um.

Figure 29. Ratio of the number of GnRH cells with BDA appositions to
the total number of GnRH cells within the tenia tecta, MS, DBB/OVLT,
and cPOA on the side of the brain ipsi- or contralateral to the BDA
injection into the medial amygdala. Independent of brain region there
was a signiﬁcantly higher proportion of BDA appositions onto GnRH
neurons on the side ipsilateral to the injection (asterisk, p:0.05. On
the ipsilateral side, cPOA had a higher BDA-GnRH ratio compared to
the tenia tecta, MS, and DBB/OVLT (b is different from a, (p50.05).

Figure 30. Four GnRH close appositions (gray arrows) onto a single
GnRH neuron in the tenia tecta. Bar, 10 um.

Figure 31. Mean GnRH-GnRH ratios (both ispi - and contralateral
sides combined) in the tenia tecta, MS, DBB/OVLT, and cPOA. The
proportion of GnRH cells with one or more GnRH appositions was
signiﬁcantly greater in the tenia tecta and MS as compared to the
DBB/OVLT and cPOA (a is different from b, ps0.05).

Figure 32. Three closely apposed GnRH cell bodies (gray arrows)
within the tenia tecta. Bar, 10 um.

xii

Figure 33. Drawing of a sagittal view of the Syrian hamster brain
(adapted from Morin and Wood, 2001) summarizing differences in
pubertal, testosterone, or chemosensory regulation of GnRH protein
(detectable cell #), GnRH mRNA in, or neuronal projections
(appositions) to the various GnRH subpopulations.

xiii

LIST OF TABLES

Table 1. Mean (:l:SEM) number of GnRH mRNA expressing cells.

Table 2. Mean body weight :I: SEM and mean seminal vesicle weight =b
SEM of juveniles and adults treated with different doses of
testosterone. Within each physiological measure, identical letters
indicate no differences between groups, whereas different letters
indicate signiﬁcant differences between groups.

Table 3. Sampling periods and swab exposure treatments of animals
used to generate time response curves.

Table 4. Mean (:hSEM) number of GnRH mRNA expressing cells 0, 60,
and 120 minutes after exposure to a swab containing pheromone.

Table 5. Summarized data from the experiments described in this
dissertation and in Parﬁtt, Thompson, Richardson, Romeo, and Sisk,
1999, asterisks). Arrows indicate the direction of change in GnRH

protein or mRNA (“’ represents no change). Checks indicate

appositions from the medial amygdala or GnRH ﬁbers onto GnRH
neurons. A higher number of checks reﬂects a higher proportion of
appositions.

xiv

KEY TO ABBREVIATIONS:

Abbmviatign

3V

ACo
AHA
ANOVA
BDA

BM
BNST
BNSTpm

CC
Ce
CPM
CV
cPOA
DBB
FSH
GABA
GnRH
GnRH+
HPG axis
HRP
xv
IM
ICC
1P
ISSH
LV
LH
NMDA
MeA
MeP
MPN
MS

Turn or Phrase

third ventricle

anterior cortical nucleus of the amygdala
amygdalohippocampal area
analysis of variance

biotinylated dextran amine
basolateral nucleus of the amygdala
bed nucleus of the stria terminalis
posteriomedial subdivision of the bed nucleus of
the stria terminalis

corpus callosum

central amygdala

counts per minute

coefﬁcient of varience

caudal preoptic area

diagonal band of Broca

follicle stimulating hormone
gamma-aminobutyric acid
gonadotropin releasing hormone
GnRH-immunopositive (-immunoreactive)
hypothalamic-pituitary-gonadal axis
horseradish peroxidase
intracerebroventricular injection
intercalalated mass cell group
immunocytochemistry
intraperitoneal injection

in situ hybridization histochemistry
lateral ventricle

luteinizing hormone
N-methyl-D,L-aspartate

anterior medial amygdala

posterior medial amygdala

medial preoptic nucleus

medial septum

XV

oc
ot
OVLT
PBS
PBS-TX
pir
PLCo
PMCo
PVP

RPA
5C
Si
SEM
SSC

TBS

optic chiasm

optic tract

organum vasculosum of the lamina terminalis
phosphate buffered saline

PBS containing 0.2% Triton-X

piriform cortex

posterolateral cortical amygdaloid nucleus
posteromedial cortical amygdaloid nucleus
polyvinal pyrolidone with heparin
radioimmunoassay

RNAse protection assay

subcutaneous injection

substantia innominata

standard error of the mean

Na/Cl/Na citrate

tenia tecta

tris buffered saline

xvi

CHAPTER 1:

GENERAL INTRODUCTION

GonadotrOpin releasing hormone (GnRH, also known as
luteinizing releasing hormone, or LHRl-l) is the primary endocrine
regulator of the reproductive system in mammalian species. Despite
their small number (less than 2000 in mammals) and scattered
distribution within the forebrain, GnRH-producing neurons are critical
to reproductive health. They ultimately determine not only the level of
gonadal steroids circulating in the blood but also whether sperm is
produced in males or ovulation occurs in females. Clinical conditions in
which the GnRH system develops abnormally, such as Kallmann's
syndrome (reviewed in Rugarli, 1999), result in failure to undergo
puberty and complete reproductive shutdown.

Hypotha/amic-Pituitary— Gonadal Axis

GnRH cells govern reproductive status through the
hypothalamic-pltuitary-gonadal (HPG) axis (Figure 1). GnRH is
released in a pulsatile manner into the median eminence of the
hypothalamus and travels through a portal system to the anterior

pituitary, where it induces pulsatile release of the gonadotropins,

 

Rostral -——— 1- Caudal

 

Gonadotropins

Estrogen o ’ ,' (LH and FSH)

Progesterone Testosterone

 

 

0“
453.11%

Gametogenesis
Steroidogenesis

 

 

 

 

Figure 1. Diagram of the hypothalamic-pituitary gonadal (HPG) axis.
follicle stimulating hormone (FSH) and luteinizing hormone (LH). FSH
and LH stimulate gamete production and maturation and steroid
hormone production and release.

Circulating gonadal steroid hormones, in turn, regulate the HPG
axis by negative feedback in males and by both negative and positive
steroid hormone feedback in females (Kalra and Kalra, 1989;Freeman,
1994). In males, high levels of testosterone resulting from increased
activation of the HPG-axis inhibit GnRH and LH secretion by action at
both the hypothalamic and pituitary levels. In females, both estrogen
and progesterone serve to inhibit HPG axis activity during the luteal
phase of the estrous (rodents) or menstrual (primates) cycle.
However, the high levels of estrogen present in the late follicular phase
exert positive feedback to induce the LH surge and subsequent
ovulation.

Development and Distribution of the GnRH system

The ontogeny of the GnRH neuronal system is unique from most
other cellular systems of the brain in that these cells originally arise in
the nasal placode (SchwanzeI-Fukuda and Pfaff, 1989;Wray, Nieburgs,
and Elkabes, 1989;Daikoku-Ishido, Okamura, Yanaihara, and Daikoku,
1990;Ronnekleiv and Resko, 1990;Silverman, Livne, and Witkin,
1994). As gestational development ensues, GnRH cells migrate along
the nervus terminalis, enter the brain, and eventually come to reside

in a number of different locations throughout the forebrain and

diencephalon. In rodent species the majority of cells are situated in
the more rostral regions such as the medial septum (MS) diagonal
band of Broca (DBB), and organum vasculosum of the lamina
terminalis (OVLT), whereas in carnivores and primates GnRH cells are
more numerous caudal to the optic chiasm (preoptic area,
hypothalamus, arcuate nucleus). The majority of GnRH neurons
project caudally to the median eminence of the hypothalamus (Jennes
and Stumpf, 1980;Silverman et al., 1994). Thus, the hormonal signal
received by the anterior pituitary reﬂects integrated neurosecretory
activity of spatially and possibly functionally diverse populations of
cells.
GnRH and Puberty

By early postnatal life, GnRH cells have come to reside in their
permanent anatomical location, their axons have innervated the
median eminence, and their cellular machinery is fully capable of
producing the mature decapeptide form of GnRH, yet these cells
remain relatively quiescent in terms of neuosecretory activity until
puberty onset (reviewed in Plant, 1994;0jeda and Urbanski,
1994;Foster, 1994). Increased secretion of GnRH at puberty, through

its regulation of the HPG axis, begins a cascade of events that

ultimately lead to maturation of the endocrine and nervous system.
Factors Impacting the GnRH System

Puberty onset results in a dramatic change in GnRH secretion,
but this developmental step is not the only factor that inﬂuences GnRH
activity. As mentioned previously, gonadal steroids are strong internal
regulators of the GnRH system. Other internal and external
(environmental) cues also provide meaningful information about
whether conditions are optimal for reproductive success. Because the
GnRH system ultimately governs reproductive status, these cells must
respond to all of these signaling systems. For example, photoperiod
(Sisk and Turek, 1983a ;Ronchi, Krey, and Pfaff, 1992b;Porkka-
Heiskanen, Khoshaba, Scarbrough, Urban, Vitaterna, Levine, Turek,
and Horton, 1997;Bernard, Abuav-Nussbaum, Horton, and Turek,
1999;Terasawa and Fernandez, 2001), pheromones (Meredith,
1991;Wysocki and Lepri, 1991;Romeo, Parﬁtt, Richardson, and Sisk,
1998), and nutritional cues (Foster and Olster, 1985 ;Berriman, Wade,
and Blaustein, 1992 ;I'Anson, Terry, Lehman, and Foster, 1997) all
inﬂuence the GnRH system. Details on how these different stimuli
regulate GnRH cells remain largely unknown.

Understanding how GnRH cells are regulated poses an

interesting neurobiological problem. The GnRH system is comprised of
a relatively small number of neurons that have a widespread
distribution. While these cells are regulated by many variables and
neurosecretory activity changes with development, investigations are
limited by the intractability of the system. Many of the standard
techniques of neuroscience (e.g., lesions, microinjections of
pharmacological agents, electrophysiology) cannot be readily used to
investigate the GnRH system, given its diffuse distribution and small
number of cells. These methodological limitations have left
neuroendocrinologists with many unanswered questions, despite years
of research.

One approach to understanding the GnRH system is to use its
unique characteristics as tools, rather than limitations, in study design.
By studying the various subpopulations of the GnRH system, one might
gain information and insight into the functional organization of the
entire system. Questions include how the different subpopulations of
GnRH neurons are regulated by so many factors and how is
information integrated within the system? Are all GnRH cells regulated
by all endogenous and exogenous signals? Or does their

responsiveness to different regulators depend on where they are

located in the brain? The local environment in which different
subpopulations of GnRH cells reside varies with neuroanatomical
location, suggesting that specialization among the different populations
is probable. More empirical evidence is needed, however, to validate
whether these different GnRH subpopulations are functionally
specialized. The following set of experiments investigates how the
GnRH system is organized, i.e., do various populations respond
differently to developmental changes such as puberty, or to regulation
by internal stimuli (e.g., steroids) or external stimuli (e.g.,
pheromones)? These experiments will determine whether testosterone
or female chemosensory stimuli impact expression of GnRH mRNA or
protein in a brain region-dependent manner. Tract tracing is also used
to address whether neurons in the medial amygdala, a chemosensory
integrating nucleus, project to GnRH neurons and, if so, whether this

neuronal input is unique to certain populations of GnRH cells.

Animal Model
The male Syrian hamster (Mesocricetus auratus) is an ideal
model species for addressing questions about GnRH subpopulation

organization and regulation for several reasons: 1) the neuroendocrine

and behavioral components of puberty have been well-established in
this animal model (Romeo, Richardson, and Sisk, 2002); 2)
distribution of the GnRH system has been described (Jennes et al.,
1980;Lehman and Silverman, 1988); 3) steroid hormones are less
potent inhibitors of LH secretion in adult male than in juvenile male
hamsters (Sisk et al., 1983a), which suggests a differential response of
the GnRH system to steroid negative feedback before and after
puberty. Therefore, we can use pubertal status as tool to investigate
testosterone regulation of GnRH neurons; and 4) GnRH is known to
play a critical role in the display of male sexual behavior, and female
chemosensory cues elicit an elevation in testosterone in male Syrian
hamsters (Meredith, 1998). Investigating how pheromones affect the
GnRH system will aid in the understanding of how information about
the external environment is transduced within the nervous system to
impact reproductive hormone secretion and behavior. Therefore, this
animal model can be used to investigate differential regulation of GnRH
subpopulations during puberty and by internal (steroid hormones) and
external (chemosensory) stimuli.

Overview of Chapters

The experiment described in Chapter 2 was designed to identify

which populations of GnRH cells likely contribute to the increased LH
release associated with pubertal maturation. To address this,
immunocytochemistry was used to identify and map out GnRH
containing cells before and after puberty and to investigate whether
particular subpopulations were associated with the onset of puberty.
This experiment is published in Brain Research (Richardson, Romeo,
and Sisk, 1999).

The experiments described in Chapter 3 and 4 were designed to
investigate testosterone negative feedback upon the different
subpopulations of the GnRH system. In these experiments, pubertal
status was included as a variable to explore whether pubertal change
in responsiveness of the HPG axis to steroid negative feedback
involves differential regulation of GnRH mRNA by testosterone before
and after puberty. In the experiment described in Chapter 3, in situ
hybridization histochemistry was used to investigate these questions.
This experiment is In press in Journal of Neuroendocrinology
(Richardson, Parﬁtt, Thompson, and Sisk, 2002). As a follow up to the
experiment described in Chapter 3, an experiment (Chapter 4) was
designed to address similar questions using a more quantitative

approach to mRNA analysis, RNAse protection assay (RPA). GnRH

mRNA and plasma LH dose-response curves to testosterone were
determined in pre- and postpubertal males to assess whether there
was differential regulation of GnRH mRNA and plasma LH between
juveniles and adults. These data will soon be submitted to
Endocrinology for publication.

Chapters 5 and 6 present experiments designed to investigate
how chemosensory information impacts the GnRH system in adults.
Female pheromones are essential chemosensory stimuli for the
expression of reproductive behavior in the male Syrian hamster. If
processing of this sensory information is curtailed, for example by
removing the vomeronasal organ or olfactory bulbs in the male, he
will not engage in mating behavior. At least part of this chemosensory
processing is thought to involve the GnRH system. Exposure to
chemosensory infomation causes a rise in plasma LH (Graham and
Desjardins, 1980;Coquelin, Clancy, Macrides, Noble, and Gorski, 1984)
and testosterone (Macrides, Bartke, Fernandez, and D'Angelo,
1974;Wirsig-Wiechmann, 1993;Romeo et al., 1998). Furthermore, icv
administration of GnRH reinstates behavior in vomeronasalectomized
males (Meredith and Howard, 1992;Fernandez-Fewell and Meredith,

1995). Thus, pheromonal information must reach and impact the

10

GnRH system through some mechanism. Experiments in Chapters 5
and 6 investigated how this external sensory stimulus impacts GnRH
cells. The ﬁrst experiment in Chapter 5 was conducted to generate a
time course for the plasma LH response to pheromone exposure in
adult animals, which has not been well-documented in the literature.
The second experiment in this chapter investigated whether female
pheromones affect GnRH mRNA in a brain region-dependent manner,
using in situ hybridization to measure GnRH mRNA.

The experiment in Chapter 6 addressed whether a
chemosensory processing region of the amygdala directly projects to
GnRH cells in any of the various subpopulations. The neural circuitry
underlying chemosensory processing and male sexual behavior has
been well-documented (Gomez and Newman, 1992;Wood and Coolen,
1997;Coolen and Wood, 1998). The medial amygdala is important for
chemosensory and processing and has been shown to project to some
of the regions in which GnRH neurons reside (Coolen et al., 1998).
Based on this knowledge, the experiment presented in Chapter 6 was
designed to investigate whether the medial amygdala projects to the
GnRH system, and if so, whether projections are speciﬁc to particular

populations of GnRH neurons. Anterograde tract tracing was utilized

11

to answer this question.
Chapter 7 is a general discussion of the questions and various
outcomes of these experiments. Data are brieﬂy summarized, and this

is succeeded by a discussion encompassing this entire body of work.

12

CHAPTER 2:
DISTRIBUTION OF GnRH NEURONS AND PUBERTAL

CHANGES IN IMMUNOREACTIVITY

Introduction

Puberty onset is associated with increased activity of the GnRH
system (reviewed in (Plant, 1994;0jeda et al., 1994;Foster, 1994).
The neural events underlying puberty activation of the GnRH system
are still being elucidated, and probably involve both decreased
inhibition and increased excitation of the GnRH system to become
active at puberty. The importance of activation occurring at this point
in development cannot be stressed enough. These maturational
processes not only induce reproductive fertility though activation of the
HPG axis, but the steroid hormonal events associated with this
increased activity are critical to pubertal maturation of the nervous
system.

With respect to the various subpopulations of GnRH cells, it is
not known if puberty arises from activation of the entire GnRH system
as a whole, or if there is some regional speciﬁcity to this

developmental change in activity. Immunocytochemical studies

13

provide evidence for a division of labor among the spatially diverse
subpopulations of GnRH cell bodies. There are several reports of brain
region-speciﬁc reductions in GnRH immunopositive (GnRH+) cell
number under conditions in which GnRH secretion is increased
(Shivers, Harlan, Morrel, and Pfaff, 1983;King, Kugel, Zahniser,
Wooledge, Damassa, and Alexsavich, 1987 ;Tang and Sisk,
1992;Ronchi, Aoki, Krey, and Pfaff, 1992a;Rubin and King,
1994;1'Anson et al., 1997). In general, these results have been
interpreted as evidence for involvement of speciﬁc subpopulations of
GnRH neurons in which somal stores of GnRH become undetectable
when secretory activity is high. For example, I’Anson et al (I'Anson et
al., 1997) demonstrated a higher number of GnRH+cells within the
medial basal hypothalamus in diet-restricted prepubertal female lambs
compared with well-fed prepubertal female lambs. The lower GnRH+
cell number in the medial basal hypothalamus of normal growing
lambs suggests that the increase in LH pulse frequency characteristic
of these lambs (Foster et al., 1985) is mediated by this particular
population of cells. Similarly, there is a brain region-speciﬁc change in
GnRH+ cell number in the arcuate nucleus of adult male ferrets

compared with prepubertal males (Tang, Kashon, and Sisk, 1997),

14

suggesting that in this species the pubertal increase in GnRH may be
primarily mediated by GnRH cells located in the arcuate nucleus.

The purpose of this experiment was to test the hypothesis that
speciﬁc populations of GnRH cells mediate the pubertal increase in
gonadotropin secretion in male Syrian hamsters.
Immunocytochemistry was used to identify GnRH cells in the brains of
adult and prepubertal males. Two different concentrations of primary
antiserum were used that were expected to result in detection of
different numbers of GnRH+ cells, depending on the somal level of
GnRH within these cells. This hypothesis predicted that when the
tissue was run with the lower (less sensitive) concentration of primary
antiserum, a reduced number of GnRH+ neurons in the adult males in

subpopulations contributing to the pubertal increase in GnRH release.

Methods
Animals

The male Syrian hamsters (Mesocricetus auratus) used in this
study were bred at Michigan State University (E. Lansing, MI) and
treated in accordance with the NIH Guide for the Care and Use of

Laboratory Animals. Protocols were approved by the MSU All-

15

University Committee for Animal Use and Care. Following weaning,
they were singly housed in a 14 hr light/10 hr dark schedule in a
temperature-controlled (21 i: 2° C) room with ad libitum access to
rodent chow (Teklad Rodent Diet No. 8640, Harlan, Madison, WI) and
water.
Study Design

At either 28 (juvenile; n=5) or 49 (adult; n=5) days of age
animals were weighed and given an overdose of Equithesin anesthetic
(80 mg/kg, ip). A cardiac blood sample was taken and plasma was
stored at -20° C until the radioimmunoassay was performed. Paired
testis and seminal vesicle weights were obtained. Animals were then
perfused intracardially with buffered saline followed by 0.4%
glutaraldehyde in 2% paraformaldehyde. The brains were stored in
20% sucrose before sectioning into consecutive sets of 40 pm thick
coronal sections. Brain sections were stored in cryprotectant at -20° C
until the immunocytochemistry was performed.
Testosterone Radioimmunoassa y

Plasma concentrations of testosterone were measured using the
Coat-A-Count Total Testosterone Kit (Diagnostic Products, Los Angeles,

CA). This assay has been validated in our laboratory for the Syrian

16

hamster. The lower limit of detectability was 0.1 ng/ml. The intra-
assay coefﬁcient of variation (CV) was 14%.

GnRH Immunocytochemistry

Qilgtign Trials:

Several different dilutions (concentrations ranging from 1:7,000-
1:80,000) of LR-1 anti-GnRH antiserum (obtained from R. Benoit,
Montreal General Hospital) were used in pilot trials to determine the
conditions under which the experimental tissue was run. LR-1 binds to
amino acid residues 2-4 and 7-10 of GnRH, and recognizes both the
mature decapeptide and the preprohormone forms of GnRH. A
concentration of 1:40,000 resulted in a range of GnRH+ staining
intensities within a single brain and was therefore chosen for the low
concentration set of experimental tissue. Concentrations lower than
1:40,000 resulted in extremely light staining, which made accurate
microscopic analyses difﬁcult. A concentration of 1:10,000 was chosen
for the high concentration set of experimental tissue because it
resulted in darker cell body staining than with the 1:40,000
concentration, without signiﬁcantly increased background staining.
Concentrations higher than 1:10,000 produced elevated background

levels, which made accurate cell counting difﬁcult.

17

Exgrimental Tissue:

Every fourth section from each experimental brain was
processed with LR-1 at a concentration of 1:10,000 and an adjacent
set of sections was processed with LR-1 at 1:40,000, in two separate
ICC runs. Within a concentration, all tissue from both age groups was
processed simultaneously. Sections were rinsed ﬁve times in 0.1 M
phosphate buffered saline (PBS), followed by ﬁve rinses in PBS
containing 0.2% Triton-X (PBS-TX). The sections were then placed in
0.3% Hzoz/MeOH at room temperature for 30 min to reduce
endogenous peroxidase activity, followed by three rinses in PBS-TX
and a 30 min incubation in normal horse serum (room temperature,
Vectastain ABC Rabbit Kit, Burlingame, CA). This incubation was
followed by 3 rinses in PBS-TX and a 24 hr incubation at 4° C in LR-1
(at either 1:10,000 or 1:40,000) diluted in PBS-TX. Sections were
rinsed 3 times in PBS-TX and incubated at room temperature for 2 hr
in biotinylated secondary antiserum (1:200, Vectastain ABC Rabbit Kit,
Burlingame, CA). This was followed by three rinses in PBS-TX and a 1
hr incubation at room temperature in an avidin-biotin horseradish
peroxidase complex (1:100, Vectastain ABC Rabbit Kit, Burlingame,

CA). Sections were then rinsed twice in PBS-TX, once in 0.05 M tris

18

buffered saline (TBS), and ﬁnally incubated in 1% 3,3'-
diaminobenzidine containing 0.0025% H202 in TBS for 15 min at room
temperature. Sections were then rinsed once in TBS, ﬁve times in
PBS, and placed in vials containing dHZO until they were mounted onto
gel-coated slides. Mounted sections were dehydrated in increasing
concentrations of alcohol, cleared in Hemo-De (Fisher, Pittsburgh, PA),
and coverslipped.
m n inin ntr

Speciﬁcity of GnRH+ staining was determined using three
controls. The ﬁrst two controls eliminated either the primary or
secondary antiserum. No staining occurred under either of these two
conditions. In the third control, the primary antiserum (1:10,000) was
preadsorbed with lug/ml of synthetic GnRH (Sigma, St. Louis, MD) at
4’C for 24 hr. While preadsorption eliminated GnRH+ staining in the
MS, DBB/OVLT, and cPOA, staining remained lateral to the DBB/OVLT
in both juvenile and adult males. This staining was not found near the
MS or cPOA GnRH cell populations. There were several differences
between the cells remaining after preadsorption (termed ambiguous
cells) and GnRH+ cells. First, nearly all of the ambiguous cells resided

lateral and somewhat ventral to GnRH+ cell populations in the

19

DBB/OVLT. These cells were generally found around the olfactory
tubercle. In addition, these cells displayed poor staining quality in
comparison to the GnRH+ cells. While GnRH+ cell body staining was
punctate, staining in the ambiguous cells was hazy and light, making
the cells difﬁcult to focus. The ambiguous cells also had remarkably
large nuclei and were often multipolar (Figure 28), whereas the
GnRH+ cells had smaller nuclei and were unipolar or bipolar (Figure
2C). Finally, the ambiguous cells did not stain with the HU4H GnRH
antiserum (mouse anti-GnRH obtained from Dr. Henryk Urbanski,
Oregon Heath Sciences Center, used at 1:2000 with the Vectastain
ABC mouse kit) nor did they express GnRH mRNA (Parﬁtt, Thompson,
Richardson, Romeo, and Sisk, 1999), further indicating that they were
not GnRH producing cells. Any cells with the characteristics of the
ambiguous cells were not counted in the microscopic analysis.
Microscopic Analysis

One experimenter blind to the condition of the animals was
responsible for all of the microscopic analyses. Tissue was

microscopically examined under brightﬁeld illumination. Cells were

20

 

an...

 

 

 

MS

LV

170 V1 7

*— 11:
D6

1
i
t

'O

Q ﬁ I

a
*4.

fl

 

 

 

 

 

 

21

Figure 2. Camera lucida line drawings depicting the three
regions analyzed: DBB/OVLT, MS, and cPOA (A, dashed boxes).
Asterisks represent the location of GnRH+ cells in a
representative animal. Abbreviations: CC, corpus callosum; LV,
lateral ventricle; AC, anterior commissure; II, optic nerve; 3V, 3rd
ventricle; OVLT, organum vasculosum of the lamina terminalis;
OC, optic chiasm. Photomicrographs depicting
immunocytochemical staining of ambiguous cells (B, arrows) and
GnRH cells (C, arrows) using LR-1 (1:10,000). Bar, 100 um.

22

considered GnRH+ if they contained brown reaction product in the
cytoplasm, were unipolar or bipolar in shape, and displayed no
characteristics of the ambiguous cells. The average number of
sections analyzed per animal did not differ between ages (15.7 :l: 1.17,
juveniles; 15.88 :l: 3.00, adults, ps0.05). Because there was variation
in the number of sections analyzed within groups (due to problems
with tissue processing), GnRH+ cell number was expressed in
GnRH+cells/section. A regional analysis was performed on the number
of GnRH+ cells/section in three operationally deﬁned areas: MS,
DBB/OVLT, and cPOA, which together contained more than 90% of the
total GnRH+ cells. Although the landmark-based boxes used to
delineate and standardize analysis of each area (Figure 2A) extended
beyond the Nissl-based boundaries of the areas, they were termed as
such because the majority of GnRH+ cells within these boxes resided
within these anatomical structures. One experimenter blind to the
condition of the animals was responsible for all of the microscopic
analyses.
Statistics

Group differences in plasma testosterone concentration and

testis and seminal vesicle weights were analyzed by two-tailed ttests.

23

Anatomical data were analyzed using a mixed design two factor
analysis of variance [age (independent factor) X antiserum dilution
(repeated measure)]. Signiﬁcant interactions and main effects were
probed using Tukey HSD and Fisher’s PLSD tests, respectively.
Differences were considered signiﬁcant if ps0.05. All data are
presented as mean :l: SEM. Due to poor tissue quality, one adult was

eliminated from the microsc0pic and statistical analyses.

Results
Peripheral Measures

Adult males had signiﬁcantly heavier paired testis (2.732 g :l:
0.137 vs 0.684 g :l: 0.053, ps0.05), seminal vesicle weights (0.209 g
:l: 0.020 vs 0.028 g :l: 0.002, ps0.05), and higher circulating levels of
testosterone (2.215 ng/ml :l: 0.361 vs 0.376 ng/ml :l: 0.156, ps0.05)
compared to juveniles.
Distribution of GnRH+ Cells

GnRH+ cells were distributed throughout the forebrain. Cells
were found in regions as anterior as the medial parolfactorial area and
posterior as the arcuate nucleus. Cells were also found in the vertical

and horizontal bands of the diagonal band of Broca, medial and lateral

24

septum, organum vasculosum of the lamina terminalis, preoptic area,
and ventral medial hypothalamus. The largest number of cells was
found within the vertical band of the diagonal band of Broca, the
second largest in the MS, and the third largest within the cPOA.
Analysis of GnRH+ Cells

Within the DBB/OVLT, juveniles had more GnRH+ cells/section
compared with adults (Figure 3A, ps0.05). There was no effect of
antiserum dilution and no interaction in the DBB/OVLT. Within the MS,
there was an interaction between age and antiserum dilution (Figure
38, ps0.05). Speciﬁcally, juveniles had more GnRH+ cells/section
than adults only under the 1:40,000 antiserum dilution condition. No
effects of age or antiserum dilution on GnRH+ cells/section were found

in the cPOA (Figure 3C).

Discussion

The present study demonstrates a region-speciﬁc decrease in
GnRH+ cell number associated with pubertal maturation in the male
Syrian hamster. Adults have fewer GnRH+ cells in the DBB/OVLT and
MS than juveniles, but the number of GnRH+ cells within the cPOA

does not differ between adults and juveniles. The most likely

25

 

 

10

 

2.

l

 

MS DPrepubertal
8- .Adult

 

 

1Oloss/OVLT
8

MAG)

GnRH-I- Cells/Section

 

 

I
5)]

 

Qt

cPOA

wedge?

 

 

1:40,000

 

Ci:-

 

1:10,000

Concentration of
GnRH antisera (LR-1)

 

Figure 3. Mean (:l:SEM) number of GnRH+ cells/section in the MS
(A), DBB/OVLT (B), and cPOA (C) of j
brain tissue was stained with a low (1:40,000) or high (1:10,000)
panel A indicate a signiﬁcant
main effect of age, with juvenile males (“a") having signiﬁcantly more
GnRH+ cells/section in the DBB/OVLT than adult males (“b")
independent of antiserum dilution (ps0.05). Letters in panel B indicate
that juvenile males (“a”) had signiﬁcantly more GnRH+ cells/section
than adult males (“b”) only with the 1:40,000 dilution of LR-l

dilution of LR-1 antiserum. Letters in

(ps0.05).

26

uvenile and adult males in which

 

explanation of these data is that fewer GnRH neurons within the
DBB/OVLT and MS are detectable in adults because increased
secretory activity of these neurons during puberty results in lower
somal levels of GnRH. Cells within the DBB/OVLT and MS of adult
hamsters could be undetectable because they no longer exist or no
longer produce GnRH in adulthood. However, the pubertal difference
in cell number was attenuated in the MS when the 1:10,000 dilution of
primary antiserum was used. Thus, it is probable that the cells within
the M5 were not detected with the 1:40,000 dilution because somal
levels of GnRH were low in these cells. Furthermore, the number of
GnRH mRNA expressing cells within the DBB/OVLT is similar in juvenile
and adults animals (Parﬁtt et al., 1999). Thus, the decrease in GnRH+
cells within the MS and DBB/OVLT in adults is in all likelihood not due
to cell death or a change in phenotype, but rather to a reduction in
GnRH somal stores resulting from increased GnRH release in
adulthood.

Cells in the cPOA did not show a pubertal reduction in number.
It is possible that cPOA cells contain higher amounts of GnRH than the
other populations and therefore remain detectable following GnRH

release. Thus, a pubertal decrease in cell number would not be found

27

in this region even though it may also play a role in increased GnRH
release in adulthood. However, in situ hybridization indicates that
GnRH cellular mRNA is not signiﬁcantly higher in cPOA cells than in the
MS and DBB/OVLT cells (Parﬁtt et al., 1999). Thus, these data only
provide evidence for the MS and DBB/OVLT GnRH neurons being
involved in the pubertal increase in GnRH secretion.

In contrast to the present ﬁndings, Urbanski et al. (Urbanski,
Doan, and Pierce, 1991) did not ﬁnd a pubertal decrease in GnRH+ cell
number in male Syrian hamsters. Variations in experimental method
could account for the difference between studies. For example, they
used a different antiserum (HU4H) than that used in the current study
(LR-1) recognizes only the mature decapeptide form of GnRH, whereas
LR-1 recognizes both the mature and the preprohormone forms. In
addition, the immunocytochemical conditions used in Urbanski et al.
(Urbanski, Doan, Pierce, Fahrenbach, and Collins, 1992) may have
been sensitive enough to detect low somal stores of GnRH.

In female hamsters, many GnRH neurons in the caudal POA
express Fos-immunoreactivity (Fos-ir), a marker of neuronal activity
(Morgan and Curran, 1991), across the entire 4-day estrous cycle. On

the other hand, cells within the DB8 and MS/rostral POA express Fos-ir

28

only on day 4, just following the preovulatory LH surge (Berriman et
al., 1992). In another report, an increase in Fos-lr was observed in
the OVLT GnRH cells just following the LH surge in female hamsters
(Doan and Urbanski, 1994). Taken together, the two studies suggest
that rostral GnRH cells are recruited for the preovulatory LH surge in
females. These data from female hamsters and the present data from
males suggest that rostral populations of GnRH neurons in the hamster
are more likely to be involved in steroid-dependent dynamic changes
in GnRH secretion than are caudal populations. The reduction in
responsiveness to testosterone negative feedback, which occurs during
pubertal maturation in male hamsters (Sisk et al., 1983a), may result
in increased release primarily from GnRH neurons residing in the MS
and DBB/OVLT.

In summary, we have demonstrated a brain region-speciﬁc
decrease in the number of GnRH+ neurons in the male Syrian hamster
as a result of pubertal maturation. These data suggest that
populations of GnRH cells in the MS and DBB/OVLT play an integral
role in the pubertal increase in GnRH release. Furthermore, these data
support the idea of inherent differences in regional populations of

GnRH neurons, raising interesting questions about when and how

29

conditions dictate a change in GnRH release. Thus, GnRH neurons in
particular anatomical locations may be responsive to regulation by

certain stimuli, such as gonadal steroids, while other populations may

not.

30

CHAPTER 3:

DOES TESTOSTERONE REGULATE GnRH mRNA?

Introduction

The last chapter provides evidence for heterogeneity among cells
within the GnRH system. In general, the more rostral populations of
cells showed a reduction in immunoreactive cell number with puberty,
indicating that they are involved in the pubertal increase in
gonadotropin release. In situ hybrdization data from our lab also
demonstrated an pubertal increase in GnRH mRNA in all GnRH
subpopulations, but it was more pronounced in the DBB/OVLT. These
data together suggest an important role of the more rostral GnRH cells
in puberty.

The threshold for steroid negative feedback regulation of the
HPG axis varies with reproductive status. Responsiveness to negative
feedback is particularly high prior to puberty in many species. For
example, in hamsters, when testosterone is experimentally clamped at
physiological levels in juvenile males, gonadotropin secretion is
completely suppressed. However, as the males enter puberty, plasma

gonadotropin levels rise despite the constantly maintained levels of

31

testosterone (Sisk et al., 1983a). Thus, a change in the negative
feedback set point is involved in the pubertal rise in gonadotropin
secretion in hamsters.

The cellular mechanisms underlying negative feedback inhibition
of GnRH neuronal activity are not well understood, nor is it known if
they change during development. One reason for this lack of
understanding is related to the diffuse distribution of GnRH neurons
throughout the forebrain, a feature that makes it difﬁcult to study the
system as a whole. Since GnRH neuronal phenotype and afferents
vary with brain region (Hoffman, Lee, Attardi, Yann, and Fltzsimmons,
1990;Wu, Segal, Miller, Gibson, and Silverman, 1992;Mitchell, Bouret,
Prevot, Jennes, and Beauvillain, 1999;Prevot, Bouret, Crolx, Takumi,
Jennes, Mitchell, and Beauvillain, 2000), the cellular level at which
steroids regulate GnRH neurons may also vary with brain region.

Regulation of GnRH mRNA is one cellular level at steroids exert
feedback effects on the HPG axis. The effects of steroids on GnRH
mRNA are brain region-dependent (Selmanoff, Shu, Petersen,
Barraclough, and Zoeller, 1991;Porkka-Heiskanen, Urban, Turek, and
Levine, 1994;Petersen, McCrone, Keller, and Shores, 1995;Spratt and

Herbison, 1997). Thus, the goals of the current experiment were two-

32

fold: 1) to conduct a brain regional analysis of testosterone regulation
of GnRH mRNA, and 2) to determine whether a change in the specific
cell groups in which GnRH mRNA is decreased by testosterone is a
correlate of the pubertal decrease in steroid negative feedback.

In some of the sections analyzed in the experiment presented in
chapter 2, an interesting phenotype of GnRH cells was found (Figure
4). These cells were quite rostral, were more clustered together, and
were embedded within a plexus of GnRH fibers. At that time, they
were considered to be the most rostral aspects of the DBB/OVLT and
MS. However, several atlases define the region in which these cells
reside as the tenia tecta. Thus, from this point on, those cells were
separated out from the other GnRH cells and analyzed as a distinct
population of GnRH neurons. These subdivisions are shown in figure 5.
Little is known about tenia tecta GnRH neurons, but they appear to be
activated by sensory stimuli to produce the preovulatory luteinizing
hormone (LH) surge in the musk shrew, a reﬂex ovulator (Dellovade
and Rissman, 1994;Dellovade, Hunter, and Rissman, 1995a;Dellovade,

Ottinger, and Rissman, 1995b).

33

 

 

 

 

 

 

 

 

Figure 4. A: Drawing of an anatomical section (adapted from Morin
and Wood, 2001) delineating the location of GnRH cells in the tenia
tecta. B: GnRH-immunoreactive cells and fibers in the tenia tecta
(see Chapter 2 for details on GnRH immunocytochemistry methods).
C: GnRH-mRNA labeled cells in the tenia tecta. Differences in
section thickness (40 pm for immunocytochemistry vs. 10 pm for
ISSH) likely account for the different number of cells in the two
photomicrographs. Bar, 20 um.

34

 

so. 3..
.383: .O.. CPOA
DEB/OVLT -

 

 

 

Figure 5. Drawing of a sagittal view of the male Syrian hamster brain
summarizing four GnRH subpopulations: tenia tecta (TT), medial
septum (MS) diagonal band of Broca/organum vasculosum of the
lamina terminalis (DBB/OVLT), and caudal preoptic area (cPOA). Red

boxes delineate the boundaries of each subpopulation.

35

In situ hybridization histochemistry was performed to measure
GnRH mRNA in the tenia tecta and MS, DBB/OVLT, and cPOA in
testosterone-treated gonadectomized juvenile and adult male Syrian
hamsters. We provide evidence for the ﬁrst time that GnRH mRNA in
neurons in the tenia tecta is reduced by testosterone in both juvenile
and adult males. While GnRH mRNA is only modestly regulated by
testosterone overall in the brain, this regulation is most pronounced in

tenia tecta neurons.

Methods
Animals, Experimental Design, and ﬁssue Collection

One day after arrival from Charles River (Kingston, NY), twelve
prepubertal (23 days old) and twelve adult (60 days old) male Syrian
hamsters (Mesocricetus auratus) were anesthetized with
methoxyﬂurane (Metofane, Mallinckrodt Veterinary Inc., Mundelein, IL,
USA), castrated, and implanted subcutaneously with either a placebo
(blank) or a 2.5 mg timed-release testosterone pellet (n=6 per
treatment group, Innovative Research, Sarasota, FL). Following
treatment, all animals were singly housed in clear polycarbonate cages

(37.5 X 33 X 17 cm) with wood chips (Aspen Chip Laboratory Bedding,

36

Warrensburg, NY). Throughout the experiment room temperature was
maintained at 21 _-l_-_ 2°C and the light-dark schedule was 14 hr light/10
hr dark (lights on at 0600 hr EST). Animals had ad libitum access to
rodent chow (Teklad Rodent Diet No. 8640, Harlan, Madison, WI) and
water throughout the study. Animals were treated in accordance with
the NIH Guide for the Care and Use of Laboratory Animals and
protocols were approved by the Michigan State University All-
Universlty Committee for Animal Use and Care.

Seven days following gonadectomy and steroid treatment (at 30
or 67 days old), animals were anesthetized with Metofane and
decapitated. Trunk blood was collected for measurement of plasma
concentrations of testosterone and LH. Brains were rapidly removed
and snap frozen in an isopentane/dry ice bath. Frozen brains were
stored at -80°C until sectioning on the cryostat. Every other coronal
section (10 um) was collected and thaw-mounted onto poly-L-lysine
coated slides to produce a total of four sets. Slides were stored with
desiccant at -80°C until in situ hybridization histochemistry was
performed.

GnRH mRNA In Situ Hybridization Histochemistry

One set of sections from each brain was processed for in situ

37

hybridization using a 35S-cRNA probe generated from Syrian hamster
GnRH cDNA (generously donated by Dr. Heiko Jansen, Washington
State Univ.). The antisense probe was transcribed in a reaction
mixture containing 1 pg of linearized DNA (BamH I linearized plasmid),
5x transcription buffer (Epicentre Technologies, Madison, WI, USA), 80
uCi [355]UT P, 120 uCi [35$]CT P, 150 uM ATP, 150 uM GT P, 12.5 mM
dithiothreitol, 20 U RNAse inhibitor, and 6 U T7 RNA polymerase
(Epicentre Technologies). Following an incubation at 37°C for 2 h,
unincorporated nucleotides were separated by Sephadex GEO-50
chromatography and the antisense probe diluted in 50% hybridization
buffer (Amresco, Solon, OH, USA) to obtain ~1.0 x 10° CPM/70 ul of
buffer. Slides were removed from the -80°C freezer and placed
immediately in 4% paraforrnaldehyde for 1 h. They were then washed
several times in 2x NaCl/Na citrate (SSC) before a 10 min incubation in
0.1 M triethanolamine (TEA) containing 0.25% acetic anhydride.
Slides were washed in dH20 and dehydrated through a series of
alcohols.

Diluted probe (70 ul) was applied onto each slide and a glass
coversllp was gently placed over the sections to prevent evaporation of

the probe during hybridization. Slides were placed in plastic boxes

38

lined with ﬁlter paper saturated with 50% formamide. The boxes were
covered with plastic lids, wrapped with plastic wrap, and incubated at
55°C for 16 h. Following hybridization, the coverslips were removed
by washes in 2x SSC and the slides were then incubated in RNAse A
buffer (200 ug/ml) for 1 h at 37°C. This incubation was followed by
several washes in decreasing concentrations of SSC (2x, 1x, 0.5x, and
0.1x) and an incubation in 0.1x SSC for 1 h at 70°C. Afterwards,
slides were washed in 0.1x SSC and dHZO rinses, dehydrated in graded
alcohols, and air-dried. Once completely dry, slides were exposed to
XAR ﬁlm (Eastman Kodak, Rochester, NY, USA) for 14 days. After
removal from ﬁlm, they were emulsion-dipped (NTBZ emulsion from
Eastman Kodak diluted 1:1 in distilled water), stored in light-tight
boxes at 4°C for 3 days, and developed using standard procedures
(Parﬁtt et al., 1999) for microscopic analysis of silver grains. Sections
were then lightly counterstained with thionin to visualize cell bodies,
dehydrated in alcohols, cleared, and coverslipped. Incubation of tissue
sections with a sense probe does not result in labeling of cells (Parﬁtt
et al., 1999).

Redeﬁning GnRH Subpopulations and Microscopic Analysis

Four populations of GnRH neurons were analyzed: tenia tecta,

39

DBB/OVLT, MS, and cPOA. These brain regions are operationally
deﬁned in Figure 6. All analyses were carried out by one investigator
blind to the treatment conditions using a Leitz Laborlux S microscope
equipped with a CCD video camera (Sony, XC-77). Labeled cells were
located under darkﬁeld microscopy at 100x or 200x magniﬁcation and
then were analyzed individually at a magniﬁcation of 400x in
brightﬁeld microc0py. Images were captured through a blue no. 47
ﬁlter used to subtract Nissl staining (Tlffen, Hauppauge, NY, USA), and
analyzed using NIH Scion Image 1.57 on a Power Macintosh 7100
computer. Each cell proﬁle was ﬁrst traced and Nissl area was
measured. After the cell proﬁle was traced and Nissl area measured, a
threshold was set so that only silver grains were visualized, and the
area (umz) covered by silver grains was measured. Although silver
grains over intensely labeled cells were not conﬁned to the Nissl
boundary, we quantiﬁed only silver grain labeling over Nissl stain
because it provided an objective deﬁnition of the cell. Thus, this
measurement is a conservative estimate of the amount of silver grain
labeling associated with the cell. The cell tracing was then moved to a

nearby region without speciﬁc hybridization to determine

40

 

 

 

 

 

 

 

 

 

 

T _
[Anterior section [W
3| : > t <
0 LV
A’l “a a I 0.
5| 0‘9 9.
'1’:
|
I
ml
2
|
|
El
>|
9..
InI
m
0|
|
(ltvll ]]
A
2| :1
o
Isis
IM
*1

 

Figure 6. Camera lucida drawings of the most anterior and posterior
sections of the four brain regions analyzed: tenia tecta, medial
septum (MS), diagonal band of Broca/organum vasculosum of the
lamina terminalis (DBB/OVLT), and caudal preoptic area (cPOA). The
area within which the GnRH cells reside is indicated by shading.
Abbreviations: II, optic nerve; 3V, 3"“l ventricle; AC, anterior
commissure; CC, corpus callosum; LV, lateral ventricle; OC, optic
chiasm.

41

background silver grain area. A GnRH-mRNA expressing cell was
deﬁned as a cell in which the area covered by silver grains was 25x
that of background. Background grain area was subtracted from the
Nissl cell grain area to obtain GnRH mRNA grain area for individual
cells. The silver grain area of all cells within each brain region was
averaged for each animal. Finally, mean silver grain area for each
region was averaged across animals within each treatment group to
obtain mean sliver grain area for the tenia tecta, MS, DBB/OVLT, and
cPOA.
Hormone Radioimmunoassays

Plasma testosterone levels were measured in duplicate samples
using the Coat—A-Count Total Testosterone Kit (Diagnostic Products,
Los Angles, CA). This radioimmunoassay (RIA) has been validated in
our laboratory for the measurement of plasma testosterone
concentrations in Syrian hamsters (Parﬁtt et al., 1999). The lower
limit of detectability was 0.1 ng/ml and the intra-assay coefﬁcient of
variation (CV) was 8.8%.

Plasma LH levels were determined by a double antibody RIA
using reference preparation RP-3 and reagents in the rat LH kit

obtained from the National Institute of Diabetes and Digestive and

42

Kidney Diseases and Dr. A.F. Parlow. To validate this assay for the
Syrian hamster, samples from intact and castrated male plasma pools
were assayed at ﬁve different dilutions. These results were compared
to a standard curve generated with the LH reference preparation
ranging from 0.8 ng to 30 ng/ml. LH values obtained from the diluted
pools were parallel to the standard curve and maintained linearity
throughout its range. All samples were run in duplicate in a single
assay. Values are reported as nanogram equivalents of NIDDK-rLH-
RP-3. The lower limit of detectability was 0.93 ng/ml and the intra-
assay coefﬁcient of variance (CV) was 13.6%.
Statistical Analysis

Two-way analysis of variance (ANOVA) was used to analyze the
effects of age and steroid treatment on plasma testosterone
concentrations, plasma LH concentrations, number of labeled cells, and
mean grain area (um2 occupied by silver grains). Signiﬁcant main
effects were probed using Fisher’s PLSD tests. The number of labeled
cells and mean grain area were analyzed separately for GnRH neurons
within the tenia tecta, DBB/OVLT, MS, and cPOA. Although each
treatment group consisted of 6 animals, actual sample sizes for the

brain regional analysis of GnRH mRNA varied because of poor tissue

43

quality resulting from occasional difﬁculties during sectioning or in situ
hybridization. When sections from a particular brain region were not
analyzable, data from that region for that animal were eliminated from
the analysis.

Because ANOVA revealed no signiﬁcant age effects, data from
adults and juveniles were combined for the frequency distribution and
regression analyses. Kolmogorov-Smimov two-sample tests were
used to determine whether frequency distributions of cells categorized
by intensity of silver grain labeling were different for testosterone-
treated and control animals within each brain region. For this analysis,
a p-value $0.001 was required for statistical signiﬁcance. Finally,
using data from individual animals in all age and treatment groups,
simple regression analyses were used to determine the correlation
between plasma LH levels and mean silver grain area in each brain
region. Except as noted above, p-values $0.05 were considered

statistically signiﬁcant.

Results

Plasma Hormone Levels

Plasma testosterone levels were undetectable (<0.1 ng/ml) in

44

juvenile and adult castrates treated with blank pellets (Figure 7, top).
At both ages, treatment with 2.5 mg testosterone resulted in plasma
hormone levels that were within adult physiological range (Sisk et al.,
1983a;Meek, Romeo, Novak, and Sisk, 1997;Parﬁtt et al., 1999).
Thus, there was a signiﬁcant increase in plasma testosterone in
castrates treated with 2.5 mg testosterone (ps0.05, Figure 7, t0p).
Plasma testosterone levels in testosterone-treated juveniles and adults
were not signiﬁcantly different.

A signiﬁcant effect of treatment on LH plasma concentrations
was found, with testosterone-treated castrates having lower plasma LH
levels than blank-treated castrates (ps0.05, Figure 7, bottom). There
was no interaction between age and testosterone on LH, indicating that
the 2.5 mg dose of testosterone reduced LH levels to a similar degree
in juvenile and adult castrates.

Number of Labeled Cells

Neither age nor hormone treatment signiﬁcantly affected the
number of labeled cells in any of the four brain regions analyzed
separately, or when regions were considered together (Table 1).

Silver Grain Analysis

Figure 8 shows labeled cells in representative adult castrates

45

 

llJuvenile *
Jiledult

    

Testosterone (ng/ml)

 

_I

 

LH(ng/ml)
w 4? 9D 09 o

 

 

 

l_

0 mg 2.5 mg
TestosteroneTreatment

Figure 7. Plasma testosterone (top panel) and LH (bottom panel)
levels in juvenile and adult males following castration and treatment
with 0 or 2.5 mg testosterone. Asterisk and bars indicate a main
effect of testosterone treatment on plasma testosterone and LH
(ps0.05) and no interaction within age. Thus, hamsters treated with
2.5 mg testosterone had significantly higher plasma testosterone than
hamsters treated with 0 mg testosterone (top panel). Hamsters
treated with 2.5 mg testosterone had significantly lower plasma LH
than hamsters treated with 0 mg testosterone (bottom panel).

46

Table 1. Mean (:l:SEM) number of GnRH mRNA expressing cells.

 

Brain Region (# of labeled cells)

 

 

Treatment
Group
Tenia DBB/ MS cPOA Total
Tecta OVLT
Juvenile 13.80 35.33 35.17 10.40 98.50
(0 mg 1337 :l:7.47 $7.55 :l:0.6 :l:9.67
testosterone)
Adult 11.60 47.40 40.20 10.80 110.00
(0 mg $2.79 :l:3.72 :l:4.47 :l:3.5 :l:11.07
testosterone)
Juvenile 9.40 31.40 26.00 8.25 77.25
(2.5 mg :l:1.89 :l:8.62 13.03 :l:1.97 5:12.20
testosterone)
Adult 15.25 41.25 31.50 7.60 95.75
(2.5 mg :l:6.05 :l:5.63 :l:4.94 :l:1.6 5:15.45
testosterone)

47

 

Figure 8. Photomicrographs of labeled cells in the tenia tecta from
castrated adults treated with 0 mg testosterone (A) or 2.5 mg
testosterone (B). Only silver grains over the Nissl stain were
measured (dark grains in plane of focus), providing a conservative
estimate of the total silver grain staining associated with each cell.

Bar, 20 um.

48

treated with either 0 (A) or 2.5 mg of testosterone (8).

Approximately 100 GnRH neurons were analyzed in each animal (all

labeled cells identiﬁed in the 1-in-8 series of sections). Mean silver
grain area values for each animal were averaged within a treatment
group for the statistical analyses. There was no effect of age or
testosterone treatment on mean silver grain area when all brain
regions were analyzed together (juvenile 0 mg testosterone, 6.96 :l:
0.79 umz; adult 0 mg testosterone, 6.73 :l: 0.56 umz; juvenile 2.5 mg
testosterone, 5.81 :l: 0.76 pmz; adult 2.5 mg testosterone, 5.06 :l:
0.64 umz). When brain regions were analyzed separately, there was a
signiﬁcant reduction in mean silver grain area with testosterone
treatment only in the tenia tecta (Ps0.05, Figure 9, top panel). This
effect was independent of pubertal status. Smaller and non-signiﬁcant
trends toward decreased silver grain area in testosterone-treated
castrates were observed in the DBB/OVLT, MS, and cPOA (Figure 9,
bottom three panels).

Because there was neither an effect of age nor an interaction
between age and treatment on area covered by silver grains, labeled
cells from juveniles and adults were pooled to create a frequency

distribution based on mean silver grain area. In the tenia tecta and

49

 

Tenia Teda

 

 

 

 

 

Mean Grain Area (pmz)
onsmm8onbmm8onhmm8o~¢am8

 

 

 

 

 

 

n=11
n=10

0 mg 2.5 mg
Testosterone

Figure 9. Mean grain area (umz) in the tenia tecta, DBB/OVLT, MS,
and cPOA in castrates (collapsed across age) treated with 0 or 2.5 mg
of testosterone. Asterisk indicates a significant effect of treatment
only in the tenia tecta (ps0.05). Testosterone treatment significantly
reduced GnRH mRNA mean grain area within this group of GnRH
neurons.

50

M5, the frequency distributions for vehicle- and testosterone-treated
groups were signiﬁcantly different, indicating an increase in the
number of lightly labeled cells (or a decrease in the number of heavily
labeled cells) with testosterone treatment (Figure 10, Ps0.001). The
frequency distributions of cells within the DBB/OVLT and cPOA did not
differ for testosterone and blank-treated animals.
Regression Analyses

The relationship between plasma LH and GnRH mRNA in each
brain region was examined by simple regression analyses of data from
individuals in all treatment/age groups. There was a modest, but
signiﬁcant, positive correlation between plasma LH levels and mean
silver grain area in the tenia tecta (Ps0.05, Figure 11, top panel). In
contrast, LH levels and mean grain area were not correlated in

DBB/OVLT, MS, or cPOA (Figure 11, bottom three panels).

51

 

 

 

 

Im- OngT(n=127)— 25mgT(n=1(B)I

 

 

 

 

  
    

 

Tenia Tecta
P5 .001
l ..... OngT(n=412) —25LgT(m2$)
MS P5001

 

 

Percent of GnRH cells

 

1“" 0 "91(rF449)— 25 rig T(n=@)|
DBB/OVLT

 

 

25

20

 

15
. ,5.
10 ’ i

b OngT(n=106)— 25n'gT(n=71)]
cPOA n,s_

 

.A
’1

 

1

Figure 10. Frequency distribution of the percentage of GnRH neurons
within the tenia tecta, DBB/OVLT, MS, and cPOA in each category of
GnRH mRNA grain area (umz). Mean grain area for each analyzed cell

6 11 16 21 26 31 36
Silver grain area (pm2)

was rounded to the nearest whole number and the cells were

categorized into bins ranging from 1 um2-36 umz. The total number
of cells for each treatment group determined n. Data from both

juveniles and adults were included in the treatment groups.

Frequency distributions for castrates and testosterone-treated males
were significantly different (Ps.001) only in the tenia tecta and MS.

52

 

 

    

 

 

 

 

 

 

 

 

1032:0207 %°Tenia 1032:0002 . ° MS
0
8' P50050 o Tecta 8 o o
i i O
6‘ 6‘
. ‘ O O
A 4- 4‘/—;’/
- i ‘ o
g 2‘ 2 O o O
5 4 0. o o
5 ....................
E 1032:9018. ° DBBI 10142:0003 ., ° cPOA
. OVLT . °
3 8‘ o o 8 O o
m 0
6‘ 6 °
‘ o ‘ o
4 ° 0 4‘/T”£/
i O i O
2‘ o o 2' o o

o
000 o o

O
00
O
0
O

 

 

 

 

 

 

o'é'4'é'ér1'112 0'2'4'6'8'1'112
Mean Grain Area (pmz)

Figure 11. Simple regression analyses of GnRH mRNA mean grain
area (umz) and plasma LH for the tenia tecta, DBB/OVLT, MS, and
cPOA. All age and treatment groups are represented in these
analyses and each dot represents data from an individual animal.
There was a significant correlation (Ps.05) between plasma LH and
GnRH mRNA mean grain area only within the tenia tecta.

53

Discussion

This study demonstrates that testosterone treatment results in
brain region-dependent reductions in GnRH mRNA, as determined by
analysis of in situ hybridization silver grain labeling. Reduced GnRH
mRNA in the presence of testosterone was reﬂected by 1) a signiﬁcant
decrease in the area of neurons covered by silver grains in the tenia
tecta, and 2) a signiﬁcant shift toward a greater percentage of lightly
labeled cells in both tenia tecta and medial septum. In the DBB/OVLT
and cPOA, these lndices of GnRH mRNA were unaffected by
testosterone. Thus, regulation of GnRH mRNA by testosterone is most
pronounced in tenia tecta neurons, intermediate in medial septum, and
absent in DBB/OVLT and cPOA. We conclude that steroid negative
feedback regulation of the GnRH neuronal system includes modest
reductions in GnRH mRNA, particularly in rostral brain regions, but also
involves other cellular mechanisms.

These data are in agreement with earlier reports that steroid
negative feedback regulation of GnRH neurons is associated with a
decrease in GnRH mRNA (Wray, Zoeller, and Gainer, 1989;Selmanoff
et al., 1991;5pratt et al., 1997). However, it is unlikely that steroid

negative feedback is exclusively exerted at the level of GnRH mRNA

54

expression. First, the magnitude of the reduction in GnRH mRNA
induced by steroid treatment in current and previous (Spratt et al.,
1997) studies is relatively modest compared with the magnitude of the
reduction in LH secretion. Second, other studies report that castration
results in either no change in GnRH mRNA (Wiemann, Clifton, and
Steiner, 1990;Malik, Silverman, and Morrell, 1991;Ronchi et al.,
1992b) or in an increase in GnRH mRNA (Park, Park, Cho, and Kim,
1988). Factors that potentially contribute to these different ﬁndings
include mRNA detection methods, time between castration and
hormone treatment, tissue collection methods, and brain regions
analyzed. Collectively, the literature suggests that steroid negative
feedback regulation of GnRH neurons is complex, occurs at multiple
cellular levels in a brain region-dependent fashion, and is dynamic

after experimental manipulation.

In the current study, the GnRH cells most affected by
testosterone negative feedback were the tenia tecta GnRH cells. The
population of GnRH neurons in the tenia tecta has not been as
thoroughly studied as those in other forebrain and hypothalamic areas.
In female musk shrews, tenia tecta GnRH cells have been strongly

linked to the sensory-induced preovulatory gonadotropin surge evoked

55

by interactions with a male, and the number of GnRH-immunoreactive
cells in tenia tecta correlates with plasma estradiol levels (Dellovade et
al., 1994;Dellovade et al., 1995a;Dellovade et al., 1995b). The
present study is the ﬁrst to link tenia tecta neurons to steroid
regulation of gonadotropin secretion in males, as GnRH mRNA was
most robustly affected by testosterone in these cells, and also was

positively correlated with plasma LH levels.

The number of GnRH neurons in the tenia tecta of the hamster is
relatively small, only slightly larger than size of the population of GnRH
neurons in the cPOA and representing approximately 10-15% of the
total population of GnRH cells. However, a small number of cells does
not preclude an important role for them in the regulation of
gonadotropin secretion. For example, studies in which GnRH neurons
were implanted into the hypothalamus of the hypogonadal (hpg)
mutant mouse (Gibson, Krieger, Charlton, Zimmerman, Silverman, and
Perlow, 1984) demonstrate that only a few GnRH neurons are
sufﬁcient to reinstate gonadotropin secretion and reproductive fertility
(Gibson et al., 1984). In addition, a general principle of the
organization of the GnRH neuronal system is specialization with

respect to afferent input and co-localization of neuropeptides and

56

neurotransmitter receptors. For example, only 5-20% of GnRH
neurons appear to express NMDA receptors (Abbud and Smith,
1995;Gore, Wu, Rosenberg, and Roberts, 1996), and only about 30%
of GnRH neurons receive vasoactive intestinal polypeptide afferents
(van der Beek, van Oudheusden, Buijs, van der Donk, Van den Hurk,
and Wiegant, 1994). Thus, tenia tecta GnRH neurons may represent a
population of median eminence projecting cells in which steroid
negative feedback regulation occurs at the level of GnRH mRNA
expression or stability. It will be important in future studies to
characterize other phenotypic traits of this group of cells in order to
understand their role in the HPG axis or other central roles. For
example, are GnRH to GnRH contacts more prevalent within this
population of cells, as is suggested by the position of GnRH cell bodies
within a dense plexus of GnRH ﬁbers in the tenia tecta? Experiment 5

addresses this question.

We found no evidence that testosterone regulates GnRH mRNA
in different populations of cells before and after puberty. We could not
determine from this experiment whether the threshold for inhibition of
GnRH mRNA increases in parallel with the increase in threshold for

inhibition of LH following pubertal maturation. The dose of

57

testosterone used in the present experiment resulted in nearly
undetectable levels of LH in both juveniles and adults. Thus, while
high testosterone levels clearly reduce GnRH mRNA in both
prepubertal and adult males, it was not possible to learn whether
pubertal status interacts with testosterone in the regulation of GnRH
mRNA. Experiment 3 will determine whether doses of testosterone
that differentially lower LH levels in juveniles and adults also
differentially results in brain region-dependent reductions of GnRH

mRNA at these two ages.
Conclusion

Among the four populations of GnRH neurons, testosterone
treatment regulated GnRH mRNA most robustly in the tenia tecta,
which was also the only population of GnRH neurons to show a
signiﬁcant correlation between GnRH mRNA and LH levels. The more
reﬁned parcellation of GnRH neurons in the current study revealed
functional differences among GnRH cells that might have been
overlooked if tenia tecta cells had been grouped with MS and
DBB/OVLT neurons as before (Chapter 2 and Parﬁtt et al., 1999).
Additional information on neighboring cell types, neural input, and

intracellular phenotype of GnRH neurons is necessary to further

58

elucidate how steroid negative feedback regulation of GnRH neurons
varies among cells in different brain regions. The experiment in
Chapter 6 provides information about the neuronal input to the various

GnRH subpopulations.

59

CHAPTER 4:
THE INFLUENCE OF TESTOSTERONE ON GNRH mRNA

BEFORE AND AFTER PUBERTY

Introduction

The previous experiment demonstrated that testosterone
reduces GnRH mRNA, as measured by in situ hybridization, within the
tenia tecta and MS subpopulations in both juveniles and adults.
Although these data indicate that testosterone impacts similar
populations of GnRH neurons before and after puberty, it does not
address the issue of whether there is a decrease in sensitivity of the
GnRH system to steroid negative feedback upon GnRH mRNA in

adulthood. This was the focus of Chapter 4.

A pivotal event in the onset of puberty is activation of the GnRH
neuronal system (Watanabe and Terasawa, 1989;Plant, 1994;0jeda et
al., 1994;Foster, 1994;5isk, Richardson, Chappell, and Levine, 2001).
The mechanisms by which GnRH neurons are activated at puberty are
not fully understood, and may involve both an increased excltatory
tone and decreased inhibitory tone. In males, testosterone (or its

intracellular metabolites) inhibits GnRH secretion via hormonal

60

negative feedback, and feedback varies with development and
reproductive status (Sisk et al., 1983a;Sisk and Turek, 1983b;Sisk,
1987;Berglund and Sisk, 1990;Lehman, Goodman, Karsch, Jackson,
Berriman, and Jansen, 1997). In many mammalian species, including
humans, puberty is accompanied by a decrease in sensitivity (or
increased threshold) of the HPG axis to steroid negative feedback
(Gupta, Rager, Zarzycki, and Eichner, 1975;Sisk et al., 1983a;Sisk,
1987;Foster, Hassing, Mendes, Hale, Padmanabhan, Hopwood, Beitins,
Marshall, and Kelch, 1989). For example, when prepubertal hamsters
or lambs are treated with a low dose of testosterone, gonadotropin
secretion is initially attenuated to undetectable levels (Sisk et al.,
1983a;Foster et al., 1985). However, when these animals reach
puberty, gonadotropin levels rise despite the clamped levels of
testosterone. Thus, the ability of gonadal steroids to inhibit
gonadotropin release decreases with pubertal maturation.

Steroid negative feedback is exerted at both the hypothalamic
and pituitary levels (Levine, Bauer-Dantoin, Besecke, Conaghan, Legan,
Meredith, Strobl, Urban, Vogelsong, and Wolfe, 1991). Centrally,

testosterone appears to inhibit GnRH secretion, and

61

in many experimental paradigms, also reduces GnRH mRNA. It

is not clear whether the pubertal change in set point for negative
feedback involves central mechanisms, and if so, whether these
mechanisms include a decrease in the magnitude of steroid dependent

reduction of GnRH mRNA.

Our lab has studied pubertal maturation of the GnRH system in
male Syrian hamsters, a species in which pubertal and seasonal
changes in neuroendocrine and behavioral responses to steroid
hormones are well-documented (Sisk et al., 1983a ;Meek et al.,
1997;Romeo, Cook-Wiens, Richardson, and Sisk, 2001). In an early
study, a single dose of testosterone was shown to be more effective in
inhibiting LH secretion in prepubertal males than in adults (Sisk et al.,
1983a). More recent work demonstrated that GnRH mRNA increased
with puberty in male hamsters (Parﬁtt et al., 1999) and is decreased
by testosterone in a brain-region dependent manner (Chapter 3). The
goal of the current study was to determine whether a pubertal change
in testosterone inhibition of LH secretion is paralleled by a similar
change in testosterone inhibition of GnRH mRNA, and LH and GnRH
mRNA dose response curves to testosterone were compared in pre-

and post-pubertal male hamsters to address this.

62

Methods
Animals

Twenty-four prepubertal (21 days of age) and 24 adult (60 days
of age) male Syrian hamsters (Charles River, Kingston, NY) were used
in the current study. The experiment was run in two identical cohorts
that were run within two weeks of each other. Animals were housed in
clear polycarbonate cages (37.5 X 33 X 17 cm) in triads from the same
treatment group. All animals had ad libitum access to food (Teklad
Rodent Diet No. 8640, Harlan, Madison, WI) and water throughout the
study. The vlvaria were on a 14 hr light/10 hr dark light-dark schedule
(lights on at 0700 hr EST) and the temperature was maintained at 21
:l: 2 °C. All animals were treated in accordance with the NIH Guide for
the Care and Use of Laboratory Animals, and the protocols were
approved by the Michigan State University All-University Committee for

Animal Use and Care.
Surgery and ﬁssue Collection

On the day of arrival, animals were castrated under
methoxyﬂurane anesthesia and immediately implanted with 3-week

time release pellets (Innovative Research of America, Sarasota, FL)

63

containing either 0, 0.5, 1.5, or 2.5 mg of testosterone (n=6/dose and
age). Seven days after treatment, animals were weighed, rapidly
decapitated, and trunk blood samples and seminal vesicles were

collected.
Brain Tissue Dissections

Brains were immediately removed after decapitation and rapidly
dissected into three tissue blocks, which were frozen on dry ice and
stored at -80° C until RNA extraction. The tenia tecta and medial
septum subpopulations could not be easily dissected from one another.
Therefore, the three tissue blocks contained GnRH neurons in the tenia
tecta/medial septum combined (Tr/MS), diagonal band of
Broca/organum vasculosum of the lamina terminalis (DBB/OVLT) and
caudal preoptic area (cPOA). Figure 12 depicts the brain dissection
procedure. Dissections were performed by placing the brain with the
ventral surface up and coronal cuts were made at the olfactory ﬁssure
(1Sit cut), the optic chiasm (2"° cut), and just anterior to the median
eminence (3rd cut). The ﬁrst of two dissected blocks (block A) was
then placed on its caudal face and two vertical cuts were made just
lateral to the left and right arms of the anterior commissure. A

horizontal cut was made just dorsal to the anterior commissure.

64

Ventral view

 

BLQQKA.’ BLQQKB;

(rostral view)

(restral vqu) (caudal view)

  
 
 

   

ﬂip block _
over —> .

 

 

DBBIOVLT
BLOCK

 

Figure 12. Camera lucida drawings of the tissue dissection
procedure. Asterisks mark the three tissue blocks collected for the
RNAse protection assay: tenia tecta and medial septum (Tr/MS),
DBB/OVLT, and cPOA. Abbreviations: II, optic nerve; 3V, 3r°l
ventricle; AC, anterior commissure; CC, corpus callosum; LV, lateral
ventricle; OC, optic chiasm; ME, median eminence.

65

The block below this horizontal cut and residing within the vertical cuts
contained the DBB/OVLT. The block above the horizontal cut and
within the vertical cuts was removed, ﬂipped over so that the caudal
face was visible, and further dissected with vertical cuts just medial to
and a horizontal cut just dorsal to the lateral ventricle to generate the
'lT/MS block. Block B was placed with the rostral face up and two
vertical cuts were made outside the left and right sides of the lateral
ventricle. Then, a horizontal cut was made just ventral to the anterior
commissure and the tissue block below this horizontal cut and within

the vertical cuts was the POA dissection.
RNA Extraction and RNase Protection Assay

In collaboration with Dr. Andrea Gore at the Mount Sinai School
of Medicine (New York, NY), GnRH mRNA levels were measured in
tissue blocks using the following protocol. Frozen dissected tissues
were homogenized in cold Trizol (Gibco, Rockville, MD), and RNA was
extracted with chloroform and isopropanol, followed by an ethanol
wash of the RNA—containing pellet. The pellet was resuspended in 30
pl diethyl pyrocarbonate-treated (DEPC) water, and a 1 pl aliquot was
used for determination of total mRNA by spectrophotometry. Samples

were aliquoted so that each contained 30 ug of total mRNA and then

66

were concentrated in a Speedvac, and resuspended in 20 ul of
hybridization buffer (0.1 M EDTA, pH 8, 4 M guanidine thiocyanate;

ﬁnal pH 7.5) for RNase protection assay.

To detect GnRH mRNA, probe (antisense) and reference (sense)
RNAs were transcribed in vitro, using a clone containing 191 bp of the
hamster GnRH cDNA, inserted into the EcoRI site of the pCRII vector
(obtained from Dr. Heiko Jansen, Washington State University,
Pullman, WA). Standard in vitro transcription methods were used to
label the GnRH mRNA probe with 3‘zP-UTP to high speciﬁcity activity
(~1,300,000 cpm/ng) (Jakubowski, Blum, and Roberts, 1991 ;Gore and
Roberts, 1994). Cyclophilin mRNA levels were determined within the
same samples to control for gel-loading variation, using a 111-bp
clone, spanning from the PstI and anI restriction sites, and
subcloned into a Bluescript KS(+) vector (Jakubowski et al., 1991),
and labeled with 32P-UTP to low speciﬁcity activity (~60,000 cpm/ng).
Both probes were brought to a ﬁnal volume of 5 pi and added to
samples and standards (20 ul each) for a ﬁnal volume of 25 pl. For
standard curves, probes were mixed with increasing known amounts of
GnRH and cyclophilin reference RNAs. After hybridization for 18 h at

30° C, the rest of the assay was conducted as described in

67

(Jakubowski and Roberts, 1992;Gore et al., 1994). Samples were
electrophoresed on 6% polyacrylamide gels, the gels were dried, and
exposed to x-ray ﬁlm for 18-36 hr in order to produce an audiogram,
then exposed to a phosphor-imaging screen (Molecular Dynamics,
Sunnyvale, CA) for 18 hr for quantiﬁcation. The amount of
radioactivity was compared to the amount of reference RNA in the
standards (Figure 13A), and regression analysis performed to produce
a standard curve (Figure 138). The resulting regression formula was

then used to determine the absolute amount of RNA in each sample.
Hormone Radioimmunoassays

Plasma testosterone concentrations were measured in duplicate
in two different assays using the Coat-A-Count Total Testosterone Kit
(Diagnostic Products, Los Angles, CA) and sample volumes of 50 ul.
The lower limit of detectability for both assays was 0.1 ng/ml and the
intraassay coefﬁcients of variation (CV) were 12.9% and 9.2%. The
interassay CV was 11%.

Plasma LH concentrations were measured in duplicate (sample
volume, 50 ul) in a single assay using reagents in the rat LH kit
obtained from the National Institute of Diabetes, Digestive, and Kidney

Diseases (NIDDK) and Dr. A.F. Parlow. Values are reported as

68

 

GnRH Befergnge RNA

0 53 125 250500 1000
A GnRH mRNA (fg)

 

 

 

 

WW
3 . y = 12141131: + 17075
Volume r2 = 1.00

 

 

 

o l l I T T j
0 mo zoo 300400 500 600
B GnRH mRNA (fg)

 

 

 

Figure 13. GnRH mRNA RNAse protection assay reference standards
(A) and standard curve (B).

69

nanogram equivalents of NIDDK-rLH-RP-3. The lower limit of
detectability was 0.66 ng/ml and the intraassay CV was 8.6%.
Statistical Analysis

Body and seminal vesicle weights and hormone concentrations
were analyzed using two-way ANOVAs (age X testosterone treatment).
GnRH mRNA data were analyzed using a three-way mixed design
ANOVA [brain region (within subject) x testosterone treatment
(between subject) x age (between subject)]. Signiﬁcant interactions
and main effects were further probed with Tukey HSD tests and
Fisher's PLSD tests, respectively. A Chi-square analysis was used to
determine whether proportion of animals responding to testosterone
treatment with plasma LH concentrations falling below detectability
differed with age. Differences were considered signiﬁcant when

ps0.05. All data are reported as mean :l: SEM.

Results
Plasma Testosterone and Physiological Measures

There was a signiﬁcant main effect of testosterone treatment on
plasma testosterone levels (ps0.05). Testosterone treatment resulted

in dose-dependent increases in plasma testosterone in both juveniles

7O

and adults (Figure 14). There was neither an effect of age nor an
interaction, such that within each dose of testosterone juveniles and

adults had equivalent plasma testosterone levels.

Mean body weight of adults was signiﬁcantly heavier than that of
juveniles (ps0.05), but body weight was not affected by testosterone
at either age (Table 2). Testosterone treatment signiﬁcantly increased
seminal vesicle weight in both juveniles and adults in dose dependent
manner (Table 2, ps0.05). At all doses of testosterone, adults had

signiﬁcantly heavier seminal vesicle weights than juveniles.

Plasma LH

There were signiﬁcant main effects of testosterone treatment
and age on plasma LH (Figure 15, ps0.05). Even though there was
not a signiﬁcant interaction between testosterone treatment and age,
plasma LH remained relatively high in adults, even with increasing
doses of testosterone. While the 1.5 mg testosterone pellet decreased
LH by only 14% in adults (compared to 0 mg treated adults), this
same dose decreased LH by 89% in juveniles (compared to 0 mg
treated juveniles). Indeed, Chi-Square analysis revealed that there

was a signiﬁcantly higher percentage of juveniles (73%) with

71

 

0|

- o - Juvenile
‘ + Adult

Plasma
Testosterone (nglml)
1° ‘1’ t

i
l

 

 

 

 

Testosterone pellet (mg)

Figure 14. Plasma testosterone levels following testosterone
treatment. Treatment significantly increased plasma testosterone in a
dose dependent manner (ps0.05). Juveniles and adults did not differ
in plasma testosterone levels with any dose.

72

Table 2. Mean body weight :l: SEM and mean seminal vesicle weight :1:
SEM of juveniles and adults treated with different doses of
testosterone. Within each physiological measure, identical letters
indicate no differences between groups, whereas different letters
indicate signiﬁcant differences between groups.

 

 

Age Dose of Body Weight Seminal Vesicle
Testosterone (9) Weight (mg)

Juvenile 0 mg 71 :l: 3° 0.021 :l: 0.001°
0.5 mg 71 :l: 3° 0.055 :l: 0.009b

1.5 mg 74 :l: 1° 0.091 :l: 0.008c

2.5 mg 72 :l: 1a 0.096 :l: 0.006c

Adult 0 mg 124 4: 1b 0.108 :l: 0.010d
0.5 mg 125 :1: 2° 0.231 :l: 0012"

1.5 mg 128 :l: 4" 0.225 at 0.026°

2.5 mg 121 1- 2° 0.263 :1: 0.027°

73

 

.5
N

- 0- Juvenile

 

 

 

 

€113 +Adult
B i
58-:
I :
.1 o-
E :
on 45
2 :
a. .
2- .‘
:———————————-—————————-—3()ﬁﬁﬂrrrr ——————
0' . . . .
o 0.5 1.5 2.5

Testosterone pellet (mg)

Figure 15. Plasma LH levels following treatment with different doses
of testosterone. Plasma LH was significantly reduced by testosterone
treatment in a dose dependent manner (ps0.05). Adults had
significantly higher plasma LH than juveniles (ps0.05).

74

undetectable levels of plasma LH than adults (29%) following
testosterone treatment (0.5 mg, 1.5, and 2.5 doses combined, X2 =
5.811, df= 1, ps0.05).

GnRH mRNA

Representative GnRH mRNA hybridization signals in “IT/MS,
DBB/OVLT, and cPOA from individual animals are shown in Figure 16.
Quantiﬁed group means are shown in Figure 17. Three-way ANOVA
(region X age X testosterone dose) revealed both region x age and
region x dose interactions on mean GnRH mRNA levels (ps0.05).

Since the effects of both testosterone and age were dependent on
brain region, these interactions were further analyzed by performing
separate two-way ANOVAs (age X dose) for each brain region.
Signiﬁcant main effects of age and testosterone on GnRH mRNA were
observed only within the TT/MS dissection (Figure 17, top panel,
ps0.05). GnRH mRNA was signiﬁcantly higher in adults and was
signiﬁcantly lowered by testosterone. Although there was not a
signiﬁcant interaction between age and treatment, testosterone
reduced TT/MS GnRH mRNA in a dose-dependent manner in adults and
did not appear to systematically affect GnRH mRNA in juveniles (Figure

17, top panel). There was no affect of age or treatment on

75

 

 

IILM§

Juvenﬂe

if

.Aduﬂ

 

 

23.519141
Juvenﬂe
lAduh

W m we «e?

W

Juvenﬂe

 

 

s44

lAduu

 

 

 

0 OJS‘LS 2A5
Testosterone pellet (mg)

 

 

 

Figure 16. Representative examples of GnRH mRNA levels in the
TT/MS, DBB/OVLT, and cPOA of gonadectomized juvenile and adult
males exposed to 0, 0.5, 1.5, or 2.5 mg testosterone pellets.

76

 

 

- 0- Juvenile
TTIMS —.— Adult

  

 

 

 

fg GnRH mRNA! ,ugRNA
.- n u s on o N

 

 

 

 

 

 

o 015 1.5 2.5
Testosterone pellet (mg)

 

 

 

Figure 17. Mean GnRH mRNA levels in the TT/MS, DBB/OVLT, and
cPOA following treatment with testosterone. Age and treatment
effects were significant only in the TT/MS (p50.05). Within the TT/MS
adults had higher mean levels of GnRH mRNA and testosterone
treatment reduced mean levels of GnRH mRNA in a dose dependent
manner.

77

GnRH mRNA levels in the DBB/OVLT or cPOA (Figure 17, bottom two

panels).

Discussion

The current study demonstrates that both testosterone and
pubertal status signiﬁcantly affected both LH secretion and GnRH
mRNA in rostral brain regions. Interestingly, however, testosterone
inhibition of LH was more pronounced in juveniles, whereas
testosterone inhibition of GnRH mRNA was more pronounced in adults.
Thus the pubertal decrease in negative feedback regulation of
gonadotropin secretion does not appear to involve a decrease in

testosterone inhibition of GnRH mRNA.

Although ANOVA did not reveal a statistically signiﬁcant
interaction between age and treatment on LH, this was likely due to
the variance in terminal LH samples inherent in the pulsatile nature of
LH secretion. Not withstanding, it is clear from the LH dose response
curves to testosterone treatment that juveniles were more sensitive to
negative feedback upon LH than adults, since the two highest doses
reduced LH to undetectable levels in juveniles, yet had little impact on

LH in adults. In fact, Chi Square analysis showed that a higher

78

proportion of juveniles, than adults, respond to testosterone with
undetectable levels of LH. This corresponds well with earlier data (Sisk
et al., 1983a), in which the same amount of testosterone treatment
inhibited plasma LH release to a greater degree before puberty than
after. Thus, increased gonadotropin release in adulthood may be
attributable to decreased sensitivity of the HPG axis to steroid negative
feedback sensitivity. Whether this change in sensitivity is occurring at
the level of the pituitary (a change in responsiveness of gonadotropes
to GnRH) or brain (increased GnRH release that is not reﬂected by

increased GnRH mRNA), or both, is not known.

GnRH mRNA in the 'lT/MS was signiﬁcantly affected by pubertal
status and testosterone treatment. Several interesting points arise
from these results. First, in the absence of testosterone, adults have
2.5 times higher GnRH mRNA in the Tr/MS than juveniles. Second,
testosterone reduces GnRH mRNA in the 'lT/MS in a dose-dependent
manner, and this reduction appears to be more robust in adults than in
juveniles. Together these two ﬁndings indicate that in adults, and to a
lesser extent in juveniles, there is tonic inhibition of GnRH mRNA by
testosterone. Furthermore, since removal of steroid negative feedback

and subsequent testosterone replacement results in no change in

79

GnRH mRNA in the DBB/OVLT or cPOA in juveniles or adults, it can
also be assumed that there is not tonic inhibition of GnRH mRNA by
gonadal hormones in these brain regions at either age. Finally, the
fact that adults are not less sensitive to testosterone negative
feedback regulation of GnRH mRNA in the TT/MS than juveniles argues
against a decreased ability of testosterone to suppress GnRH mRNA as

an explanation for the pubertal rise in gonadotropin release.

Since GnRH neuronal phenotype and afferents vary with brain
region (Hoffman et al., 1990;Wu et al., 1992;Mitchell et al.,
1999;Prevot et al., 2000), it is not surprising that puberty and
testosterone have a differential impact on GnRH mRNA across the
regional subpopulations of GnRH neurons. Brain region-dependent
changes in GnRH mRNA in response to puberty and testosterone
negative feedback are consistent with data published from this and
other laboratories (Chapter 3;Spratt et al., 1997;Tang et al., 1997).
In the current study these two variables affected GnRH mRNA
exclusively in the dissection that included the tenia tecta and MS GnRH
neurons. This ﬁnding corresponds well with previous results from the
in situ hybridization experiment (Chapter 3), in which GnRH mRNA

was most robustly reduced by testosterone in the tenia tecta, and to a

80

lesser extent in MS. Furthermore, testosterone did not affect in situ
hybridization signal of GnRH mRNA in the DBB/OVLT or cPOA. Since
GnRH neurons in all brain regions project to the median eminence, we
assume that they all release GnRH to regulate gonadotropin secretion.
Yet, two different methods of quantifying GnRH mRNA lead to the
conclusion that GnRH mRNA is most highly regulated in rostral
populations of GnRH neurons and that overall, ﬂuctuations in GnRH
mRNA do not predict ﬂuctuations in LH secretion. Thus, there may be
heterogeneity among GnRH neurons in the relationship between
mRNA, protein, and release.

In summary, these data demonstrate a pubertal increase in
plasma LH and in GnRH mRNA levels in the tenia tecta and medial
septum. The pubertal increase in LH secretion appears to depend on a
decrease in sensitivity to steroid negative feedback. Puberty results in
decreased sensitivity to testosterone inhibition of plasma LH, but this is
not accompanied by a decreased sensitivity to testosterone inhibition
of GnRH mRNA. Thus, although testosterone attenuated GnRH mRNA
in the TT/MS, the ability of testosterone to do so was not enhanced in
juveniles. While these two components of the HPG axis clearly

undergo changes in response to puberty and testosterone, they do not

81

appear to be directly related to one another. Instead, the
developmental shift in the set point of negative feedback regulation of
gonadotropin secretion may permit increased activity of the GnRH
system at puberty, an event that is occurring simultaneously, but

possibly through unrelated, steroid-independent mechanisms.

82

CHAPTER 5:

PHEROMONES AND THE GnRH SYSTEM

Introduction

In many species, including humans (Jacob, Hayreh, and
McClintock, 2001), various social cues pertinent to reproduction are
conveyed to the brain through the vomeronasal and olfactory systems.
In rodents, urine or vaginal secretions from the female contain
chemosensory stimuli that have a powerful inﬂuence over the
reproductive neuroendocrine system and behavior in the male
(Meredith, 1998). Presenting a receptive female to the male induces
gonadal steroid release and initiates mating behavior from the male.
GnRH is thought to play a critical role in these responses (Meredith,
1998). Interruption of chemosensory processing (e.g.,
vomeronasalectomy) blocks both the rise in androgens and the
behavioral response to females (Wysocki et al., 1991;Meredith et al.,
1992 ;Pfeiffer and Johnston, 1994), and central microinjection of GnRH
reinstates behavior in these animals (Meredith et al., 1992 ;Femandez-

Fewell et al., 1995).

The neuroendocrine response to female pheromones has been

83

investigated in rodent species. In male Syrian hamsters, exposure to
female pheromones causes a rise in plasma testosterone 30-60 min
later (Macrides et al., 1974;Wirsig-Wiechmann, 1993;Pfeiffer et al.,
1994;Romeo et al., 1998). This is assumed to occur because of an
elevation in plasma LH secondary to increased release of GnRH.
However, while an elevation in LH following exposure to female urine
has been documented in mice (Coquelin et al., 1984) and rats
(Graham et al., 1980), evidence for such a response in hamsters is
lacking. Unpublished studies from our lab have consistently
demonstrated statistically non-signiﬁcant trends toward elevated
plasma LH following exposure of males to vaginal secretions from
females. The most probable reason for an ambiguous LH response is
that the pulsatile nature of LH results in inherent individual variability,
which potentially precludes detection of group differences in mean LH
in terminal blood samples. In addition, the timing and amplitude of
the LH response may vary across animals. One way to resolve this
issue is to obtain frequent blood samples from individual animals
following exposure to pheromones, which allows for comparison of

pheromone-induced changes in plasma LH within an animal.

Evidence for GnRH’s role in behavior comes from research in

84

which icv administration of GnRH reversed the impairment of male
sexual behavior brought about by vomeronasalectomy (Wysocki et al.,
1991 ;Meredith et al., 1992;Pfeiffer et al., 1994;Femandez-Fewell et
al., 1995). GnRH has also been used to condition a place preference in
males (de Beun et al., 1991), suggesting that it may have rewarding
properties and be involved in motivation aspects of behavior. These
studies argue for GnRH’s involvement in chemosensory processing and
behavior, but do not provide answers to how the sensory information
is relayed to and regulates the GnRH system. Attempts to utilize c-fos
expression as a marker of neuronal activity in GnRH neurons in males
have not been successful, and therefore have not provided answers to

questions (Meredith, 1998).

The goals of the current study were two-fold: 1) to document
and characterize the LH response of male hamsters to female
pheromones and 2) to investigate whether pheromone exposure
results in brain region-dependent increases in GnRH mRNA. In order
to address these questions, two separate experiments were carried out
using adult male Syrian hamsters. First, animals were ﬁtted with
jugular catheters in order to obtain serial blood samples prior to and

following exposure to cotton swabs containing female pheromones or

85

control cotton swabs. Second, animals were presented with female
pheromones, and at 0, 60, or 120 min after exposure brains were
collected for in situ hybridization analysis of GnRH mRNA within the

tenia tecta, MS, DBB/OVLT, and cPOA.

Methods

Experiment 1: Time course of LH response to female pheromones in

adult males.
Animals

Fourteen sexually naive adult male Syrian hamsters (Charles
River, Kingston, NY) were used in this study. Animals were singly-
housed in clear polycarbonate cages (30.5 X 20.5 X 30.5 cm). All
animals had ad libitum access to food (Teklad Rodent Diet No. 8640,
Harlan, Madison, WI) and water throughout the study. The vivaria
were on a 14 hr light/10 hr dark light-dark schedule (lights on at 0600
hr EST) and the temperature was maintained at 21 :l: 2 °C. All animals
were treated in accordance with the NIH Guide for the Care and Use of
Laboratory Animals, and the protocols were approved by the Michigan

State University All-University Committee for Animal Use and Care.

86

Jugular Catheterizations

Cateterization and blood sampling protocols were modiﬁcations
of the procedures used in (Gillespie, van der Beek, Mintz, Mickley,
Jasnow, Huhman, and Albers, 1999) for repeated blood sampling.
Under sodium pentobarbital anesthesia (80 mg/kg, ip), animals were
ﬁtted with catheters into the right jugular vein. Catheters were made
from 7 cm lengths of silastic tubing (Dow Corning, Midland, MI,
od=0.037 in id=0.020 in), and a silastic cuff was secured with a small
amount of super glue 2.5-2.8 cm from the proximal (animal) end of
the catheter, which was bevel-cut for easier insertion into the jugular
vein. A piece of L-shaped metal tubing (22 guage) was attached to the
opposite end of the tubing. The catheter was inserted into a small
incision within the jugular vein and held in place by suture ties on
either side of the cuff. The entire catheter was subcutaneous, except
the metal portion, which exited from animal’s back, at the nape of the
neck (secured in place by wound clips and super glue). This metal
connector allowed for the animal to be easily hooked up to a long piece
of polyethylene tubing (od=0.038 in ik0.023 in, Becton Dickinson
and Company, Sparks, MD) on the day of blood sampling, so that he

would not have to be tethered after surgery and could freely move

87

around his home cage throughout the experiment. In between surgery
and blood sampling, the distal 2-3 cm portion of the catheter was ﬁlled
with a viscous heparinized solution containing Polyvinal Pyrrolidone
(PVP, mol wt 40,000 Sigma, St. Louis, MO), and the metal connector
was capped with a short piece of PE tubing closed off at one end. PVP
helped keep the catheter patent so that the animals could be allowed a

few days recovery after surgery before blood sampling.
Blood Sampling and Experimental Design

On the day of blood sampling, the animal was brieﬂy restrained,
the polyethylene cap was removed, and a long piece of polythene
tubing ﬁlled with heparinized sterile saline was attached to the metal
connector. This tubing was connected to 22 gauge blunted needle on
a 3 cc syringe, which was used to draw up the PVP followed by a small
amount of blood. The volume drawn out was replaced with
heparinized saline (0.3-0.5 cc).

Blood sampling began 15-45 min later (~2-3 hr before lights out,
1300). The animal remained in his home cage throughout the
sampling procedure. Four baseline samples were taken (5 min apart,
0.2 cc blood/sample) and a cotton swab (containing either vaginal

secretions from estrous female hamsters or left dry) was presented

88

during the 5th baseline sample. Blood sampling continued at the same
rate (0.2 cc, every 5 min) until 30 min after swab exposure. Three
more samples were taken at 15 min intervals so that the last sample
was withdrawn 75 min after the swab was ﬁrst presented to the
animal, and 95 min after baseline blood sampling began. Throughout
the sampling period, the animal was given back his own red blood cells
(diluted with heparinized saline), which were obtained following
centrifugation and removal of plasma from earlier samples. Pilot work

demonstrated that blood replacement did not alter plasma LH levels.

Following the sampling period, the animal was brieﬂy restrained,
the tubing was removed, a small amount of PVP was injected into the
catheter, and the PE cap was replaced. When possible, animals were
used twice for blood sampling (allowing at least 4 days between
sampling). In an attempt to randomize experience, animals were
exposed to either pheromone swabs for both sampling periods, control
swabs for both sampling periods, a pheromone swab for the 1St and a
control swab for the 2"‘1 sampling period, or a control swab for the 1St
and a pheromone swab for the 2'” sampling period. Of the 14 animals,
8 had patent catheters and could be used for blood sampling. One of

these 8 was eliminated from the study because blood samples weren’t

89

obtainable throughout the entire sampling period and another was
eliminated because he died a day after the sampling, indicating that he
might have been sick during the sampling period. Of the remaining 6
animals, 3 were sampled twice and 3 sampled from once for a total of 9
sampling periods (5 pheromone swab exposure and 4 blank swab
exposure, Table 3) that could used to generate time response curves.

Plasma I_H Radioimmunoassays

Plasma LH levels were determined in single samples for in two
assays using the methods described in Chapter 3. The intra-assay CVs
were 8.7% and 13.1% and the inter-assay CV was 11.5%. The

minimum detectable levels of LH were 0.38 ng/ml and 0.39 ng/ml.
Data Analyses

The data from each sampling period were plotted as percent
change from baseline. Baseline was determined for each sampling
period by averaging the 3”, 4*", and 5‘" baseline samples (or 2 of these,
when 3 were not available). The percent change from baseline was
then calculated for each sample after this point, for each individual
profile. Since data were not independent (e.g., two sampling periods
generated from the same animal), non-parametric statistics were used

for the analyses. Separate Kruskal-Wallis tests were used to

90

Table 3. Sampling periods and swab exposure treatments of animals

used to generate time response curves.

 

 

Animal 15" Sampling 2'“l sampling
95 blank ---
104 blank ---
105 blank blank
594 pheromone pheromone
595 pheromone pheromone
976 pheromone —

91

investigate plasma LH changes across time following exposure to
pheromone and following exposure to control swabs. A Mann-Whitney
U test was used to compare plasma LH levels following exposure to
pheromone versus control swabs, collapsed across time. Differences

were considered statistically signiﬁcant when ps0.05.

Experiment 2: Pheromonal regulation of GnRH mRNA in adult males.
Animals

Fifteen sexually naive adult male Syrian hamsters were obtained
from Charles River (Charles River, Kingston, NY). Animals were singly-
housed in clear polycarbonate cages (37.5 X 33 X 17 cm. All animals
had ad libitum access to food (Teklad Rodent Diet No. 8640, Harlan,
Madison, WI) and water throughout the study. The vivaria were on a
14 hr light/10 hr dark light-dark schedule (lights on at 2300 hr EST)
and the temperature was maintained at 21 :l: 2 °C. All animals were
treated in accordance with the NIH Guide for the Care and Use of
Laboratory Animals, and the protocols were approved by the Michigan

State University All-University Committee for Animal Use and Care.
Experimental Design

Between 0900 and 1200 cotton swabs containing vaginal

92

secretions from estrous females were dropped into animals’ cages.
Animals were killed by decapitation at 0 (no swab dropped), 60, and
120 minutes after exposure to the pheromone containing swab
(n=5/time point). The actual clock time of exposure was counter-
balanced across animals from the different groups in order to control
for the effects of circadian time on plasma LH and testosterone.
Terminal blood samples were obtained for measurement of LH and
testosterone by radioimmunoassay, and brains were collected, snap-
frozen on dry ice, and stored at -80°C until sectioning on the cryostat.
Every other coronal section (10 um) was collected and thaw-mounted
onto poly-L-lysine coated slides to produce a total of four sets. Slides
were stored with desiccant at -80°C until in situ hybridization

histochemistry was performed.
GnRH mRNA In Situ Hybridization Histochemistry

One set of sections from each brain was processed for in situ

hybridization using the protocol described in Chapter 3.
Microsc0pic Analysis
All analyses were carried out by one investigator blind to the

treatment conditions using a protocol similar to that described in

Chapter 3. However, in addition to obtaining the area of silver grains

93

covering the Nissl-deﬁned cell body covered by silver grains (“cell
grain area"), the area of the entire cluster of silver grains associated
with a particular cell (which sometimes extended beyond the Nissl-
deﬁned cell body; “spread grain area”) was also measured in the
current study. Both analyses were performed in order to determine if
the pattern of results was similar when more (cell grain area) or less
(spread grain area) conservative estimates of labeling were used.
Thus, three variables were measured in each animal in the current
study: number of labeled cells, cell grain area, and spread grain area

within the tenia tecta, MS, DBB/OVLT, and cPOA.
Plasma Hormone Radioimmunoassays

Plasma LH levels were measured in duplicate in a single assay,
using the methods described in Chapter 3. The intra-assay CV was
12.4%. The lowest detectable level of LH was 0.73 ng/ml.

Plasma testosterone levels were measured in duplicate samples
using the methods described in Chapter 3. The intra-assay CV was
16.4% and minimum detectable was 0.1 ng/ml.

Data Analyses
Mean number of labeled cells, mean cell grain area, and mean

spread grain area for the tenia tecta, MS, DBB/OVLT, and cPOA were

94

compared across the different time points using separate one-way
ANOVAs for each brain region. Data were also analyzed by combining
data across brain regions. Plasma LH and testosterone levels were
compared between the different groups using one-way ANOVAs.
Signiﬁcant main effects were probed using Fisher’s PLSD tests.

Differences were considered signiﬁcant when ps0.05.

Results

Exgriment 1: Time course of LH response to female pheromones in

adult males.

Examples of individual LH proﬁles following exposure to
pheromone versus control swabs are shown in Figure 18. There was a
rise in plasma LH that began around 10 min after exposure to
pheromone (Figure 18 A). This rise was seen in all animals to which
pheromones were presented, although individual responses differed
slightly with respect to the magnitude and timing of elevations in LH
across sampling periods. Plasma LH levels remained elevated until 25-
30 minutes alter the pheromone-containing swab was ﬁrst introduced.

Plasma LH levels did not rise when animals were exposed

95

 

 

 

 

 

 

 

 

 

 

 

 

A
3.
24
E 1-
2’ 4

I t i ' ' I I I ' i I I I '
" 4

g . o = pheromone (#595) B

3 0 = blank (#104)

n. 3-
21

1- ¢ /

. e— ‘_ -
.50'4'0'5'1'0'50'50 45 so 75

Time Since Exposure to Swab (min)

Figure 18. Representative example plasma LH proﬁles following
exposure to a cotton swab containing pheromone (A) or a control swab
(B). The arrows indicate delivery of the swab (pheromone=black
arrow, blank=clear arrow).

96

to control swabs (Figure 18 8). Figure 19 shows sampling periods,
which are grouped according to swab exposure and expressed as %
change from baseline. Nonparametric analyses revealed a signiﬁcant
change in plasma LH from baseline across time in response to
pheromone exposure (ps0.05). Exposure to control swabs did not
result in signiﬁcant changes across time. Overall, there was a greater
change from baseline LH levels with pheromone exposure, as

compared to control swab exposure (ps0.05).
Experiment 2: Pheromonal regulation of GnRH mRNA in adult males
Plasma Hormone Levels

There was not a signiﬁcant effect of time after pheromone
exposure on plasma LH levels (Figure 20). However, there was a
signiﬁcant effect of time after pheromone exposure on plasma
testosterone (Figure 21, ps0.05). Plasma testosterone was
signiﬁcantly higher at 60 min as compared to 0 min and 120 min after

exposure to pheromone.
Number of Labeled Cells

Pheromone exposure did not signiﬁcantly affect the number of
labeled cells in any of the four brain regions, either when analyzed

separately or when regions were considered together (Table 4).

97

 

+Pheromone
1 50, .. 'O'Blank

100‘

 

 

 

 

 

Plasma LH (% change from baseline)

 

 

 

'5 10152025550 7 ' 4'5' ' so '75
Time since exposure to swab (min)

Figure 19. Time response curves of mean (:l: SEM) plasma LH (%
change from baseline) following exposure to pheromone or blank
swabs. There was a signiﬁcant increase in plasma LH only after
exposure to pheromone (p50.05).

98

 

Plasma LH (nglml)

 

 

 

0 60 120
Time since exposure to swab (min)

Figure 20. Mean (:l: SEM) plasma LH levels (ng/ml) 0, 60, and 120
min following exposure to swabs containing pheromone. There was
not a signiﬁcant effect of pheromone on LH across time (p>0.05).

99

 

Plasma testosterone (nglml)

 

 

 

0 60 120
Time since exposure to swab (min)

Figure 21. Mean (:l: SEM) plasma testosterone levels (ng/ml) 0, 60,
and 120 min following exposure to swabs containing pheromone.
Testosterone was signiﬁcantly elevated 60 min after exposure
(ps0.05).

100

Table 4. Mean (:tSEM) number of GnRH mRNA expressing cells 0, 60,
and 120 minutes after exposure to a swab containing pheromone.

 

 

 

Time since Brain Region (# of labeled cells)
exposure to
swab (min)
Tenia MS DBB/OVLT cPOA Total
Tecta
0 18.67 1 31.80 :l: 42.00 :h 9.00 :l: 98.67 :l:
3.84 4.57 11.00 2.30 22.21
60 9.00 :l: 38.80 :1: 46.00 :l: 9.60 :1: 102.00 :1:
2,27 4.01 8.67 5.77 14.22
120 13.60 :1: 29.20 :l: 38.60 i 6.00 d: 87.40 :l:
2.52 2.08 5.80 5.52 7.47

101

However, there was a trend (although non—significant, P=0.13) of a

reduction in the number of labeled cells within the tenia tecta.
Silver Grain Analysis

There was no effect of time following exposure to pheromone on
mean cell grain area (0 min=12.27 :l: 0.87 umz, 60 min=12.45 :l: 0.91
umz, 120 min=12.58 :l: 1.35 umz) or mean spread grain area (0
min=39.32 :l: 0.72 umz, 60 min=40.99 d: 5.85 umz, 120 min=41.00 :l:
5.36 umz) when the four subpopulations were considered together.
Figure 22 shows mean cell grain and spread grain areas separately for
the tenia tecta, MS, DBB/OVLT, and cPOA. There were no significant
effects in any of the four GnRH subpopulations of pheromone exposure
on mean cell grain area (left graphs) or mean cell spread area (right

graphs).

Discussion

These data demonstrate that chemosensory information from the
female impacts the hypothalamic-pituitary-gonadal axis at both the
level of plasma LH and testosterone in sexually naive adult male Syrian
hamsters. Pheromone exposure results in a rise in plasma LH that

peaks at about 15 minutes and a subsequent rise in testosterone by 60

102

 

 

8

18

Tenia Tecta Tenia Tecta

   

.5

B .

 

 

O)
0

MS

 

rec.
..°.‘.°..

 

 

O)
O

DBBIOVLT

DBBIOVLT

b.
O

N
9

-L-L
.'\.’.9’.¢.‘?.

Mean silver grain area over and surrounding cell (pmz)

 

 

 

Mean silver grain area over cell (pmz)
a?
O
C

18. cPOA cPOA

A
O

N
C?

 

 

 

 

   

0 so 120 ‘ 0 so 120
Time since exposure to pheromone (min)

Figure 22. Mean area of silver grains (umz) as measured within the
boundary of the cell body (left graphs) and within and surrounding the
cell body (right graphs) in the tenia tecta, MS, DBB/OVLT, and cPOA of
pheromone exposed males. Pheromones did not affect GnRH mRNA, as
measured by either index, at any time point following exposure
(p>0.05).

103

minutes after exposure. Activation of the HPG-axis by female
pheromones, however, did not result in changes in GnRH mRNA at
either 60 or 120 minutes after exposure. Thus, although pheromones
likely induce GnRH secretion to result in elevated LH, activation of
GnRH neurosecretion is not accompanied by increases in GnRH mRNA

at the time points studied.

To our knowledge, this is the first description of an LH response
to female pheromones in male Syrian hamsters. Since LH begins to
rise within 10 min after presentation of the pheromone stimulus, we
infer that activation of the GnRH system is an immediate neural
response to the stimulus (Levine and Ramirez, 1982). This timing fits
well with a proposed central role of GnRH in male reproductive
behavior, which is initiated within 30 seconds of being introduced to a

receptive female (Meek et al., 1997;Romeo et al., 2001).

The pheromone-induced rise in testosterone by 60 min
corresponds well with previous data from this lab (Romeo et al., 1998).
Based on previous studies, testosterone levels are elevated before 60
minutes (Macrides et al., 1974;Pfeiffer and Johnston, 1992;Wirsig-
Wiechmann, 1993;Pfeiffer et al., 1994). However, even if testosterone

levels are increased earlier, it is not likely that this rise in gonadal

104

steroids is necessary for reproductive behavior with the female at that
time. Since male hamsters often engage in the whole suite of
behaviors, including ejaculation, within minutes of being placed with a
receptive female, the rise in testosterone 30-60 min after exposure to
chemosensory information from the female probably does not play an
acute role in behavior, but instead, might have long-term functions
(e.g., altering neural or behavioral responses to females in future

encounters).

Pheromones did not result in changes in GnRH mRNA, as
measured by labeled cell number, mean grain cell area, or mean grain
spread area either 60 or 120 min after exposure. It could be argued
that changes in GnRH mRNA could be occurring earlier than 60 min,
since plasma LH levels are high 15 min following exposure to
pheromones. However, in a recent report, GnRH mRNA was not altered
in male mice exposed to bedding containing female urine 45 min
following exposure, even though plasma LH levels were significantly
higher than control-treated animals 45-90 min after exposure (Gore,
Wersinger, and Rissman, 2000). Thus, although pheromones clearly
affect GnRH release, as indexed by plasma LH, this response does not

result in altered levels of GnRH mRNA in these cells.

105

One important ﬁnding from these data is that assessing GnRH
mRNA by measuring silver grains within and surrounding the Nissl-
deﬁned cell body (spread grain area) provides similar trends as
measuring silver grains exclusively within the Nissl-defined body (cell
grain area). Thus, the more conservative estimate of silver grain
labeling (assessing only those cells that reside over the Nissl-defined
cell) does not account for the lack of changes in GnRH mRNA following

exposure to pheromones.

Another interesting finding is that there was a trend towards a
reduction in cell number and GnRH mean grain area in the tenia tecta
at 60 minutes, indicating that GnRH mRNA may be slightly lowered in
this cell group following pheromone exposure. On the other hand,
perhaps this is not related to pheromone-induced release of GnRH from
these cells, but rather to the high levels of testosterone at 60 min.
Testosterone reduces GnRH mRNA specifically in the tenia tecta
(Chapter 3). Perhaps the rise in testosterone 60 min following
pheromone might be high enough to initiate negative feedback

reduction of GnRH mRNA levels in these cells.

In summary, the first experiment of this chapter demonstrated

that chemosensory information from females induced a rise in plasma

106

LH from baseline 10-30 minutes after exposure that was followed by a
return to baseline levels 15 minutes later. The second experiment
demonstrated that pheromones also induced an increase in plasma
testosterone by 60 minutes following exposure. Although this hormone
response is likely reﬂecting increased GnRH release shortly after
exposure to pheromones, GnRH activation is not reflected in changes in
GnRH mRNA. Thus, either the GnRH response is not large enough to
stimulate synthesis and measurable changes in mRNA, or the timing of
such changes occurs earlier or later than 1-2 hours after exposure to

pheromones.

107

CHAPTER 6:
ANATOMICAL RELATIONSHIPS AMONG GNRH NEURONS

AND MEDIAL AMYGDALA EFFERENTS

Introduction

As stated in the last chapter, an important external signaler of
the reproductive neuroendocrine system in male Syrian hamsters is
chemosensory information from the female. Female pheromones
induce neuroendocrine (rise in LH and testosterone) and behavioral
responses from the male, and the GnRH system is thought to be
involved in both (Meredith, 1998). How chemosensory information
from the periphery reaches GnRH neurons is largely unknown. Much
research has focused on how chemosensory information reaches cell
groups comprising the behavioral circuit in male Syrian hamsters. This
experiment takes advantage of what has been established about the
neural circuitry underlying chemosensory processing and sexual
behavior in the male Syrian hamster to address questions about how

chemosensory information may reach the GnRH system.

When adult males are exposed to vaginal secretions from

females, the immediate early gene protein product Fos, a marker of

108

neuronal activation (Morgan et al., 1991), is expressed in virtually all
components of the forebrain circuit underlying male sexual behavior
(Fiber, Adames, and Swann, 1993;Fiber and Swann, 1996;Kollack-
Walker and Newman, 1997;Swann and Fiber, 1997;Romeo et al.,
1998). Stimulated brain regions include the anterior (MeA) and
posterior (MeP) portions of the medial amygdala, posteriomedial
subdivision of the bed nucleus of the stria terminalis (BNSTpm), and

medial preoptic nucleus (MPN).

The current study focused on the efferent projections of the
medial amygdala. It has already been established that the anterior
portion of the medial amygdala sends moderate projections to the
vertical (Gomez et al., 1992;Coolen et al., 1998) and horizontal
(Gomez et al., 1992) limbs of the diagonal band of Broca, lighter
projections to the medial septum (Gomez et al., 1992), and dense
projections to the medial POA (Kevetter and Winans, 1981;Maragos,
Newman, Lehman, and Powers, 1989;Gomez et al., 1992;Coolen et al.,
1998). All of these areas contain GnRH cell bodies, and chemosensory
information could be delivered to the GnRH system via any of these

routes.

This experiment was designed to address whether GnRH cells are

109

innervated by efferents from the medial amygdala. Given the diffuse
distribution of GnRH cell bodies, retrograde tract tracing cannot be
effectively used to answer questions about neural input to the GnRH
system. Instead, anterograde tract tracing was used to determine
whether efferents from the amygdala project directly to GnRH cell
bodies, and if so, whether projections were to cells within speciﬁc GnRH
subpopulations. Biotinylated dextran amine (BDA) was used as a
neuronal tract tracer in combination with GnRH ICC to determine
whether projections from medial amygdala make apparent contacts
onto GnRH cell bodies. GnRH subpopulations were analyzed separately
to determine whether the proportion of GnRH neurons receiving medial

amygdala appositions varied with brain region.

Since immunocytochemical procedures in the current study were
designed to optimize GnRH immunoreactivity, and each GnRH cell was
individually analyzed at high magnification for close appositions from
BDA—immunoreactive fibers anyway, close appositions from GnRH-
immunoreactive fibers were also assessed in these brain sections. The
close proximity of GnRH neurons in the tenia tecta to GnRH
immunoreactive ﬁbers (discussed in Chapters 2 and 3) suggests a

greater amount of GnRH-GnRH communication within this

110

subpopulation. Thus, the proportion of GnRH neurons receiving close
appositions from GnRH processes was determined and comparisons

were made across the four GnRH subpopulations.

Methods
Animals

Sexually naive adult male Syrian hamsters (Charles River,
Kingston, NY) were used in this study. Animals were group-housed
until stereotaxic surgery and were singly housed afterwards. Cages
were made of clear polycarbonate (37.5 X 33 X 17 cm) and contained
wood chips (Aspen Chip Laboratory Bedding, Warrensburg, NY). All
animals had ad libitum access to food (Teklad Rodent Diet No. 8640,
Harlan, Madison, WI) and water throughout the study. The vivaria
were on a 14 hr light/10 hr dark light-dark schedule (lights on at 0600
hr EST) and the temperature was maintained at 21 d: 2 °C. Animals
were treated in accordance with the NIH Guide for the Care and Use of
Laboratory Animals, and the protocols were approved by the Michigan

State University All-University Committee for Animal Use and Care.

111

Iontophoretic Injections

On the day of surgery, animals were anesthetized with sodium
pentobarbital (80 mg/kg). The head was shaved and positioned in a
stereotaxic apparatus (David Kopf Instruments, Tujunga, CA)and 0.03
cc of local aesthetic, lidocaine HCL 2% and epinephrine (1:10,000
Injection USP, Abbot Laboratories, North Chicago, USA) was injected
under the skin just before surgery. Biotinylated dextran amine (BDA,
10%, MW 10,000 Sigma, St. Louis, MO) was delivered through a glass
micropipette, which was pulled from Kwikfil microtubules (World
Precision, Sarasota, FL) so that the tip had an inner diameter of 10-20
pm. The micr0pipette was slowly lowered into the brain to the medial
amygdala (0.5 mm anterior and 2.8 mm lateral to bregma, and 6.5 mm
ventral to dura) alternating sides randomly among animals. BDA was
iontophoretically injected using a current source (Midguard, Stoelting,
Wood Dale, IL) that delivered +5 pA positive (cathodal) alternating
current (at 7 sec on/7 sec off intervals). After the injection, the current
was turned off and micr0pipette left in place for 10 min. A 0.1uA of
negative (anodal) current was applied as the micr0pipette was slowly
withdrawn from the head to prevent diffusion of BDA out of the

micropipette.

112

Post-Injection Survival Time

Although tract tracers generally have a predominant direction of
transport, either retrograde or anterograde, transport can go in both
directions (Lanciego, Luquin, Guillen, and Gimenez-Amaya, 1998).
Analysis after different post-injection survival times can help determine
the degree to which a tracer will result in retrograde versus
anterograde labeling (Angelucci, Clasca, and Sur, 1996;Lanciego et al.,
1998). Therefore, a pilot study was performed in which animals were
killed at 2, 4, 7, and 14 days following BDA injection in order to
determine the survival time for that resulted in the least amount of
retrograde staining, while allowing enough time anterograde transport
from the amygdala to the most rostral region of interest in this study,
the tenia tecta. A survival time of 7 days was selected for several
reasons. First, if animals were killed 2 days after the injection, BDA
was diffusely spread across the neuropil, rather than within a defined
group of cell bodies, making the center of the injection difﬁcult to
assess (Figure 23, top two panels). This was somewhat improved by 4
days, but the boundaries of the injection sites were the most distinct
following 7- and 14-day survival times. Second, retrogradely-filled

cells were found in areas outside the injection site following 4,

113

 

Figure 23. Photomicrographs showing medial amygdala BDA injection
sites in representative animals killed 2, 4, 7, and 14 days after surgery.
Survival times of 2 and 4 days resulted in diffuse spread of BDA. The
injection sites were more distinct 7 and 14 days after surgery.

114

7, and 14 day survival times. Thus, there was no advantage of
selecting a 4-day survival time given that the injection site was clearer
at 7 and 14 days. Since 7 days resulted in sufficient anterograde
transport (from the amygdala to rostral forebrain areas), this survival
time was chosen for the experiment.

Perfusion and Histology

Animals were overdosed with sodium pentobarbital and perfused
intracardially with 100 ml Buffered Saline Rinse, followed by 150 ml
4% paraformaldehyde. Brains were removed from the skull and post-
ﬁxed in 4% paraformaldehyde. Pilot work revealed that post-fixing
brains for 12 hr, as compared to 1 and 4 hr, improved
immunocytochemical staining of GnRH cell bodies and fibers, especially
in the tenia tecta and MS. Following post-ﬁxation, brains were
submerged into 30% sucrose for 24 hours. Four sets (40pm) of coronal
sections were cut using a cryostat and stored in phosphate
buffer/sodium azide at 4°C if immunocytochemistry was run within a
few weeks or in cryoprotectant at -20°C for longer storage periods.
Storage of sections did not affect immunocytochemical staining of BDA
or GnRH. One set of tissue was processed and analyzed from each

animal.

115

A double labeling immunocytochemical procedure was employed
to visualize BDA and GnRH in the same sections. A protocol was
generated using modifications of published methods described to
visualize BDA (Coolen et al., 1998) and GnRH (Chapter 2). Sections
were washed several times in 0.1 M PBS/2% Triton X-100 (PBS-TX) and
incubated in 3%H202/methanol for 30 minutes at room temperature to
block endogenous peroxidase activity. They were then rinsed with 0.1
M PBS-TX and incubated in with normal goat serum (1:67, Vectastain
Elite Kit, Vector) for 30 minutes at room temperature. Immediately
following this step, sections were submerged in vials containing LR-1
anti-GnRH primary antiserum (1:10,000 in PBS-TX, R. Benoit, Montreal

General Hospital) and incubated for 24 hours at -4° C.

Following incubation in anti-GnRH primary antiserum, sections
were processed through a series of steps to visualize BDA staining.
Sections were removed from vials containing the antisera and rinsed
several times in 0.1 M PBS, followed by an incubation in ABC-HRP,
1:800, Vectastain ABC Elite Kit, Vector) for 1 hour. Tissue was then
rinsed several times in 0.1 M PBS and the HRP was visualized with
NiCl-enhanced 3,3'-diaminobenzidine chromagen to produce a blue-

black product over BDA labeled neurons and axons. Most

116

photomicrographs in this chapter are displayed in color so that GnRH
(black) and GnRH (brown) staining can be differentiated from one

another.

After completion of this reaction, sections were taken through the
rest of the steps for visualization of GnRH. First, tissue was rinsed
several times in 0.1 M PBS and incubated at room temperature in 1%
Hzoz/PBS for 10 minutes to block unreacted HRP from the BDA
visualization steps. Sections were then rinsed several times in 0.1 M
PBS-TX and incubated for 120 minutes at room temperature in
secondary biotinylated goat-anti rabbit antiserum (1:200, Vectastain
ABC Elite Kit, Vector). This was followed by several rinses in 0.1 M
PBS-TX and a 60 min incubation at room temperature in ABC-HRP
(1:100 Vectastain ABC Elite Kit, Vector). Tissue was rinsed two times
in 0.1 M PBS-TX, once in 0.05 M Tris Buffered Saline, and HRP was
visualized with 3,3'-diaminobenzidine chromagen to produce a brown
product over GnRH labeled neurons and axons. Sections were mounted
onto glass slides, dehydrated in increasing concentrations of alcohol,
cleared in Hemo-De (Fisher, Pittsburgh, PA), and coverslipped for

microscopic analysis.

117

Microsc0pic Analysis

Eleven animals were selected for analysis of BDA and GnRH
appositions onto GnRH neurons. Nine of the animals had injection sites
that were within or encompassed the medial amygdala. Two had
injections that were on the medial side of the optic tract within the
substantia innominata (SI) and served as controls. Sections from these
brains were examined under brightfield illumination. GnRH cells were
analyzed in the tenia tecta, MS, DBB/OVLT, and cPOA. Cells were first
located and counted at 200x magnification. Each cell was then
analyzed individually at a magniﬁcation of 1000x using a 100x oil

objective and viewing through 10x eyepiece.

Since synaptic contacts onto GnRH neurons could not be
established using light microscopy, close appositions onto GnRH
neurons were assessed in the current study. The criteria for identifying
a GnRH neuron with close appositions from a GnRH process (“GnRH-
GnRH cell") were as follows. GnRH appositions most often appeared as
large brown dots (similar in size to the boutons along GnRH axons),
and less often as finer processes, onto a GnRH cell body, or dendritic or
axonal process. The GnRH fiber had to be distinguishable from the cell

body (i.e., not just a region of punctate GnRH-immunoreactive staining

118

within that particular cell), but had to appear to be contacting the
GnRH neuron at 1000x magniﬁcation. Often, processes would appear
to be in close apposition to a GnRH cell under lower magnification (e.g.,
500x), but at 1000x magnification it became evident that the process
was either above or below the cell. Thus, a 3-dimensional view of the
entire cell was obtained by adjusting the focus through the depth of the
cell, which allowed for the elimination of false positives (when the
processes appeared to be closely apposed to the cell, but actually was
just above or below it). The place in which the apposition and GnRH
neuron “contacted" had to come into focus together in the same focal
plane in order to be considered a close apposition. Each GnRH cell with
one or more close appositions from GnRH-immunoreactive processes
was counted as a single GnRH-GnRH cell.

Requirements for identifying close appositions of BDA onto GnRH
neurons were essentially the same as for GnRH appositions (e.g., the
place where the BDA apposition touched the GnRH cell had to be visible
in the same focal plane at 1000x), except the requirements were
somewhat stricter. Black dots and small lines that resided exclusively
within the boundaries of GnRH cells were not counted. The reason for a

more conservative criteria for BDA appositions is that GnRH-

119

immunoreactive staining is sometimes characterized by very dark
brown staining around the perimeter of the cell body and axons, which
is hard to distinguish from black immunoreactive staining. In order to
avoid mistaking this staining for BDA appositions, a BDA-
immunoreactive processes had to be visible approaching the GnRH cell
and also be visible in the same focal plane as the GnRH cell (at the
place of “contact”) in order to be considered a BDA close apposition.
Each GnRH cell with one or more close appositions from a BDA-

immunoreactive process was counted as a single BDA-GnRH cell.

For each animal, the number of GnRH neurons in the tenia tecta,
MS, DBB/OVLT, and cPOA was first counted on each side of the brain
(ipsi- and contralateral to the injection). The number of GnRH-GnRH
and BDA-GnRH cells was then assessed for the different subpopulations
on each side of the brain. These values were then used to determine
GnRH-GnRH and BDA-GnRH ratios. The GnRH-GnRH ratio was
calculated by dividing the number of GnRH-GnRH cells by the total
number of GnRH neurons (within a subpopulation on the ipsi or
contralateral side of the brain). The BDA-GnRH ratio was calculated by
dividing the number of BDA-GnRH cells by the total number of GnRH

neurons (within a subpopulation on the ipsi or contralateral side of the

120

brain). For statistical comparisons, mean GnRH-GnRH ratios and BDA-
GnRH ratios were determined for animals within a group (SI versus
medial amygdala injected animals). Since injection site did not alter
GnRH-GnRH ratios, mean GnRH-GnRH ratios from all animals were
combined for statistical analyses. Comparisons between mean GnRH-
GnRH ratios on the ipsi- versus contralateral side of the brain and
across GnRH subpopulations were made using a repeated measures 2
(side of brain) x 4 (brain region) ANOVA. A repeated measures 2 (side
of brain) x 4 (brain region) ANOVA was used to compare mean BDA-
GnRH ratios on the ipsi- versus contralateral side of the brain and
across GnRH subpopulations in animals with medial amygdala
injections. Few BDA-GnRH cells were found in animals with S1
injections and these animals were not included in the mean BDA-GnRH
ratio statistical comparisons. Significant main effects were probed
using Fisher’s PLSD tests. Differences were considered significant when

p50.05.

121

Results

An average of 133.78 :l: 6.84 immunoreactive GnRH cells were
analyzed for each animal. The mean number of cells varied across
areas: tenia tecta, 26.90 :l: 2.80; MS, 48.09 :l: 3.00; DBB/OVLT, 37.27

5: 3.43; and cPOA, 21.55 :1: 2.95.
BDA Appositions

There was some variation in the size and location of injection
sites, but in all cases of injections into the amygdala, a considerable
portion of the injection included the medial amygdala. Examples of two
medial amygdala injection sites (one small and one large) and one SI
injection site are shown in Figure 24. In all medial amygdala injected
animals, fiber tracts were seen connecting the medial amygdala , BNST,
and MPN, as previously described (Gomez et al., 1992;Coolen et al.,
1998). Interestingly, GnRH and BDA labeled fibers were found
traveling in separate groups, but beside one another, in the stria
terminalis (Figure 25). GnRH fibers were also found within the stria
terminalis in the two substantia innominata injected brains, but the
BDA fiber tract patterns seen in animals with medial amygdala
injections (Gomez et al., 1992;Coolen et al., 1998) were not evident in

the SI injected animals. A few GnRH processes

122

 

 

 

 

 

 

 

123

Figure 24. Schematic representations of animals with BDA
injections into the medial amygdala (A,#806; B, #809) and
substantia innominata (C, #810). Animal #806, #809, and #810
had BDA-GnRH ratios of 1.0, 0.9, and 0, respectively, in the cPOA
ipsilateral to the injection. Abbreviations: ACo, anterior cortical
nucleus of the amygdala; AHA, amygdalohippocampal area; BM,
basolateral nucleus of the amygdala; Ce, central nucleus of the
amygdala; IM, intercalcalated mass cell; MeA, anterior medial
amygdala; MeP, posterior medial amygdala group; oc, Optic
chiasm; ot, optic tract; PMCo, posteromedial cortical nucleus of
the amygdala; PLCo, posterolateral cortical nucleus of the
amygdala, si; substantia innominata.

124

 

Figure 25. GnRH and BDA ﬁbers in the stria terminalis from an animal
with a medial amygdala BDA injection. Bar, 20 pm.

125

were also found within the amygdala, and in one case, a GnRH axon
was double-labeled with BDA (presumably due to retrograde transport,
Figure 26).

In animals with injections into the medial amygdala, BDA—labeled
ﬁbers closely apposed GnRH neurons in all four brain regions. Close
appositions of BDA onto GnRH neurons were found on the cell body
(Figure 27, top), and on the dendritic or axonal (Figure 28) processes
of GnRH neurons. There was a signiﬁcantly higher proportion of BDA-
GnRH neurons in the same side of the brain as the BDA injection,
independent of brain region (Figure 29, p50.05). Few appositions were
found on the side contralateral to the injection (Figure 29). On the side
ipsilateral to the medial amgydala injection, cPOA had a higher
proportion of BDA-GnRH cells, as compared to the tenia tecta, MS, and
DBB/OVLT (Figure 29, p50.05). BDA-GnRH cells were seldom found on
either side of the brain or in any of the GnRH subpopulations in the two
animals with substantia innominata injections (data not shown).

GnRH Appositions

GnRH appositions onto GnRH neurons were evident in all brain

regions. Figure 27 (bottom) shows a GnRH neuron with two close

126

— \

Figure 26. GnRH (gray arrows) and BDA (black arrows) double labeled
fiber from an animal with a medial amygdala injection. Bar, 20 pm.

127

 

 

Figure 27. Photomicrographs of a GnRH cell in the cPOA viewed in
different focal planes from an animal with a medial amygdala BDA
injection. The black arrow indicates a BDA close apposition and gray
arrows indicate GnRH close appositions onto the same cell. Bar, 10
pm.

128

 

Figure 28. Photomicrograph of a GnRH neuron in the tenia tecta with a
BDA apposition onto the axon from an animal with a medial amygdala
BDA injection. Also note that a second GnRH cell body is in close
apposition to this GnRH neuron.‘ Bar, 10 pm.

129

 

 

 

 

    

 

 

 

 

 

1
E] Tenia tecta
b [3 MS

.8 - n DBBIOVLT
.g 1 I cPOA
CU
it .6 _ a
i 4 l
:
<9 .4 - *
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8

2 _

 

 

 

ipsilateral contralateral

Side of Brain (relative to injection)

Figure 29. Ratio of the number of GnRH cells with BDA appositions to
the total number of GnRH cells within the tenia tecta, MS, DBB/OVLT,
and cPOA on the side of the brain ipsi- or contralateral to the BDA
injection into the medial amygdala. Independent of brain region there
was a significantly higher proportion of BDA appositions onto GnRH
neurons on the side ipsilateral to the injection (asterisk, ps0.05. On
the ipsilateral side, cPOA had a higher BDA-GnRH ratio compared to the
tenia tecta, MS, and DBB/OVLT (b is different from a, (p50.05).

130

appositions onto the cell body from GnRH processes. In most cases,
GnRH neurons in the tenia tecta and MS had multiple GnRH appositions
(Figure 30), however the number of appositions per cell were not
quantiﬁed. Analysis of mean proportion of GnRH cells receiving one or
more close GnRH appositions is summarized in Figure 31. There was
no difference between the ipsi- or contralateral side of the brain in the
GnRH-GnRH ratio (p >0.05). Therefore, ipsi- and contralateral GnRH-
GnRH ratios in the tenia tecta, MS, DBB/OVLT, and cPOA were averaged
and these data are shown in Figure 31. There was a higher proportion
of GnRH-GnRH cells in the tenia tecta and MS as compared to the
DBB/OVLT and cPOA (ps0.05). There were no significant differences
between GnRH-GnRH ratios in the tenia tecta and MS (p >0.05), or
between GnRH-GnRH ratios in the DBB/OVLT and cPOA (p >0.05). An
interesting phenomenon was noted in these sections. In all brains
analyzed, a small number of GnRH cell bodies was found adjacent to
one another (Figures 28 and 32). In some cases, as many as four
GnRH cell bodies were found closely apposed. This phenomenon was
seen in all GnRH subpopulations, but to a lesser extent in the cPOA, in
which closely apposed GnRH cell bodies were found in only a few

animals.

131

 

Figure 30. Four GnRH close appositions (gray arrows) onto a single
GnRH neuron in the tenia tecta. Bar, 10 pm.

132

 

_x
o
O

A O) on
C) O O
0)

Percent of GnRH cells
receiving GnRH appositions

N

O

  

 

 

0

TT MS DBBIOVLT cPOA
Brain Region

Figure 31. Mean GnRH-GnRH ratios (both ispi - and contralateral
sides combined) in the tenia tecta, MS, DBB/OVLT, and cPOA. The
proportion of GnRH cells with one or more GnRH appositions was
significantly greater in the tenia tecta and MS as compared to the
DBB/OVLT and cPOA (a is different from b, ps0.05).

 

Figure 32. Three closely apposed GnRH cell bodies (gray arrows)
within the tenia tecta. Bar, 10 pm.

134

Discussion

The current study implicates the medial amygdala as a possible
relay station for chemosensory information to GnRH neurons.
Anterogradely labeled projections from the medial amygdala were
found in close proximity to GnRH neurons in all subpopulations, with
the largest proportion of close appositions to GnRH neurons in the
cPOA. This is the ﬁrst evidence of direct efferent projections from the
amygdala to GnRH cells, providing an a potential pathway by which
pheromonal stimuli from the female can reach the neuroendocrine

system.

Although these data demonstrate an anatomical relationship
between the medial amygdala and GnRH neurons, it remains to be
determined whether there is a synaptic relationship between medial
amygdala and GnRH neurons. Electron microscopy is one approach
that could help establish evidence of a synaptic relationship between
amygdala projections and GnRH neurons. Electron microscopic studies
have reported a relatively small number of synaptic inputs onto GnRH
cell bodies (Witkin and Silverman, 1985;Lehman et al., 1988).
However, due to the scattered distribution of GnRH neurons and

technical difﬁculties in fixing tissue for visualization of GnRH

135

immunoreactivity at both the light and electron microscopic levels
(Witkin, 1999), only a small number of neurons (e.g., 2 cells/brain,
Lehman et al., 1988) have been analyzed to generate these data.
Studies using confocal microscopy coupled with synaptophysin labeling
to investigate synaptic input to GnRH cells, which allows for the
analysis of more GnRH neurons per brain, have reported higher
estimates of synaptic input to GnRH cells (Rajendren and Gibson,
2001;Dudas and Merchenthaler, 2001 ;Rajendren and Li, 2001). Future
investigations using confocal microscopy to estimate synaptic input
from the medial amgydala to GnRH neurons would provide further
anatomical support for communication between this region of the brain

and the GnRH system.

The fact that BDA close appositions were most prevalently found
on cPOA GnRH neurons compared with other GnRH subpopulations
raises the possibility that these cells are particularly more responsive to
chemosensory stimuli. With such a small amount of information known
about how pheromones inﬂuence GnRH cells, it is of particular interest
that electrical stimulation to the vomeronasal organ (Meredith and
Fewell, 2001) elicits expression of Fos, used as an index of neuronal

activation, in GnRH neurons only in the cPOA. The importance of the

136

medial amygdala in chemosensory processing (Fiber et al., 1993;Fiber
et al., 1996;Kollack-Walker et al., 1997;Swann et al., 1997;Romeo et
al., 1998) and the fact that these cells project to GnRH neurons
predominantly in the cPOA, the "GnRH subpopulation activated by
vomeronasal organ stimulation (Meredith et al., 2001), supports the
hypothesis that pheromones influence the neuroendocrine system

through this pathway.

The medial amygdala is comprised of cells of various phenotypes,
including cells that make substance P (Swann and Newman, 1992) and
steroid receptors (Wood, Brabec, Swann, and Newman, 1992;Coolen et
al., 1998). In addition to chemosensory information, projections to the
GnRH system may be relaying other information. Perhaps the
amygdalar projections to some of the GnRH cells are coming from
androgen or estrogen receptor expressing cells. Since steroid action
upon GnRH cells is most likely indirect (Herbison, Robinson, and
Skinner, 1993;Herbison, Horvath, Naftolin, and Leranth, 1995), these
cells might serve as indirect means by which steroid could negatively
feedback upon GnRH mRNA. Information from smaller, more defined
injections, combined with steroid-receptor immunocytochemistry could

help clarify this issue.

137

In addition to projections from cells in the medial amgydala to
GnRH neurons, close appositions from GnRH fibers were also found on
GnRH neurons. The proportion of GnRH-GnRH cells was greatest in the
tenia tecta and MS. Within the tenia tecta and MS GnRH cells
frequently had many appositions from GnRH fibers. GnRH cell bodies
also found closely apposed to one another in these same populations.
These ﬁndings correspond well with the fact that these cells appear
different from other populations (especially the DBB/OVLT and cPOA
cells), in that they appear close together and ensconced in a bed of
fibers (Chapter 2). This experiment does not establish the origin of the
GnRH fibers apposing GnRH neurons or whether these appositions
contain synapses, but the findings strongly suggest some form of
communication within the GnRH system. An interesting possibility is
that there could be gap junctions between GnRH fibers and cell bodies
and between cell bodies that are closely apposed to one another. Gap
junctions have been identiﬁed in the GnRH immortalized cell line, GT-l
cells, and shown to important for synchronized pulsatile activity of
these cells (Witkin, 1999;Terasawa, 2001;Funabashi, Suyama, Uemura,
Hirose, Hirahara, and Kimura, 2001 ;Vazquez-Martinez, Shorte,

Boockfor, and Frawley, 2001). In addition, GnRH cell bodies have been

138

shown to form an intercellular bridge (Witkin, 1999). Perhaps both
synaptic and non-synaptic mechanisms underly communication within
the GnRH system, and this allows cells to be coordinated together and

send meaningful signals to the pituitary gland.

In summary, these data provide evidence of direct projections
from the medial amygdala to GnRH neurons. Cells within the cPOA had
a large proportion of BDA appositions. The data indicate possible
pathway by which chemosensory information can reach the GnRH
system to influence release and that GnRH-GnRH communication
varies depending on the brain region. Future work with more reﬁned
injections and immunocytochemical identification of the various medial
amygdala cellular pheontypes could reveal whether the same
population of medial amygdala neurons are projecting to GnRH neurons

in the tenia tecta, MS, DBB/OVLT, and cPOA.

139

CHAPTER 7:

GENERAL DISCUSSION

The overall goal of this dissertation research was to gain a better
understanding of how a unique and important set of peptidergic
neurons are organized within the brain to respond to various internal
and external stimuli. GnRH cells govern the entire reproductive system
through their regulation of the hypothalamic pituitary gonadal (HPG)
axis. While their contribution to biological function is greatly
appreciated, it is largely unknown how these cells operate to respond to
and integrate variables that determine reproductive function. The
wide-spread distribution of such a small number of neurons has

impeded a full understanding this system.

Experiments in this dissertation demonstrated that the
anatomical location of GnRH cells relates to how these cells are
regulated by internal and external stimuli, and following pubertal
maturation. These findings are summarized along with other work in
our lab (Parﬁtt et al., 1999) in Table 5 and Fig 33. Data presented
throughout the dissertation provide evidence for heterogeneity within

the GnRH system in male Syrian hamsters in response to different

140

 

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141

   
    
 

A A
GnRH-GnRH appositions

 

GnRH-GnRH appositions A
Testosterone (mRNA

Testosterone (mRNA)
Puberty (detectable
Puberty (mRNA) cell #, mRNA)

TT L .- MS
:00.

”.0 :o....- ?

331?;1 ° c P O A
,0 e e

baa/OVLT“: ' .
Chemosensory medial
Puberty (detectable cell #) amygdala appositions)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 33. Drawing of a sagittal view of the Syrian hamster (adapted
from Morin et al., 2001) brain summarizing differences in pubertal,
testosterone, or chemosensory regulation of GnRH protein (detectable
cell #), GnRH mRNA in, or neuronal projections (appositions) to the
various GnRH subpopulations.

142

factors, a finding that corresponds well with other reports (Shivers et
al., 1983;King et al., 1987;Berriman et al., 1992;Ronchi et al.,
1992a;Rubin et al., 1994;1'Anson et al., 1997;Tang et al., 1997).
There is variation among the four forebrain GnRH populations, tenia
tecta, MS, DBB/OVLT, and cPOA, under different developmental states
(before and after puberty), and in response to an strong internal
stimulus (testosterone). Furthermore, there is anatomical evidence for
subpopulation differences in potential communication within the GnRH
system and between other non-GnRH neurons within the medial
amygdala and the GnRH system. Thus, function and regulation of
GnRH neurons may be related to the location within the brain because

neuronal input to GnRH cells varies with location.

Rostral populations of GnRH neurons appear to play an important
role in puberty. There is a reduction in detectable cell number in the
DBB/OVLT and MS, which is thought to reflect increased GnRH release
from these cells. Furthermore, there is a pubertal increase in GnRH
mRNA in the tenia tecta and MS, which may also be related to GnRH
release. Because the tenia tecta GnRH subpopulation was not analyzed
as a separate population from the MS and DBBIOVLT in first study, it is

not clear whether pubertal changes in detectable cell number also

143

occurs in tenia tecta neurons, or if some of these cells contributed to
the changes shown in the MS and DBBIOVLT. Consequently,
comparisons between pubertal changes in GnRH protein and mRNA are
limited because analyses of GnRH subpopulations were different in
these two studies. Nevertheless, assuming there is some overlap
between GnRH cell groups that show pubertal changes in GnRH protein
(presumed to be reflecting increased release) and mRNA, an important
question to ask is whether the pubertal rise in GnRH mRNA precedes

the increased release of the peptide at puberty.

The relationship between GnRH synthesis and release is not well
understood. Explant cultures of neonatal hypothalmic slices have been
used to investigate regulation of GnRH mRNA (Maurer and Wray,
1997). Under these in vitro conditions, GnRH neurons have the most
rapid mRNA turnover rate (ti/2, 5-13 min) reported for neuropeptides.
The decay of mRNA corresponds well, temporally, with the descending
portion of GnRH secretory episodes measured in viva (Levine and
Ramirez, 1980;Levine et al., 1982;Dluzen and Ramirez, 1987), which
argues for a tight coupling between GnRH synthesis and release. If
these in vitro GnRH mRNA findings generalize to GnRH neurons in vivo,

increased synthesis of GnRH might precede increased levels of secreted

144

hormone at puberty. However, it is also possible that the primary
event associated with puberty is activation of GnRH release, which
leads secondarily to an increase in GnRH synthesis as the demand for
releasable hormone goes up. Thus, if there is a causal relationship
between increases in GnRH mRNA and GnRH release at puberty, the

causal direction is presently not known.

The role steroids play in increased activity of the GnRH at puberty
depends on the species. While the pubertal increase in GnRH release in
primates occurs in the absence of steroid negative feedback (Terasawa
et al., 2001), the pubertal increase in GnRH mRNA in ferrets seems to
involve changes in steroid negative feedback of the GnRH system (Tang
et al., 1997). Data presented in this dissertation provide evidence in
against a relationship between sensitivity to steroids and pubertal
changes in GnRH mRNA in male Syrian hamsters. Pubertal maturation
resulted in higher levels of GnRH mRNA in the tenia tecta and MS
(Chapter 4). In the absence of steroids adults had over two-fold higher
levels of GnRH mRNA and adults were more, not less, sensitive to
testosterone negative feedback upon GnRH mRNA. Thus, while the
pubertal increase in GnRH mRNA might be due to increased excltatory

(Petersen, McCrone, Keller, and Gardner, 1991;Gore et al., 1994) or

145

decreased inhibitory (Sim, Skynner, Pape, and Herbison, 2000) tone
upon cells within the tenia tecta and MS, steroids are clearly not driving
this process. This is different from the pubertal increase in
gonadotropin release, which involves a change in steroid negative
feedback (Chapter 4; Sisk et al., 1983a). When steroid negative
feedback is removed, plasma LH is equivalent in adults and juveniles
(Chapter 4). However, in the presence of the same dose of
testosterone, plasma LH is suppressed in juveniles, but not adults
(Chapter 4; Sisk et al., 1983a). Therefore, steroids are involved in the
rise in gonadotrOpins at puberty. While GnRH mRNA and plasma LH
undergo changes in response to puberty and testosterone, these two
components of the HPG axis do not appear to be related to one another.
Instead, the developmental shift in the set point of negative feedback
regulation of gonadotropin secretion may permit increased activity of
the GnRH system at puberty, an event that is occurring simultaneously,
but possibly through unrelated, steroid-independent mechanisms.

A dissociation between GnRH mRNA and plasma LH was also
demonstrated in Chapter 5. GnRH mRNA does not appear to be
regulated by chemosensory stimuli. Exposure to female pheromones

elicited an increase in plasma LH, but this was not accompanied by in

146

changes in GnRH mRNA. Thus, while pheromones ostensibly stimulate
GnRH release (as indexed by plasma LH release), this release is either
not great enough to result in measurable changes in GnRH mRNA using
in situ hybridization histochemistry, or message levels are changing
earlier than 60 min after exposure. However, a recent report on mice
demonstrated that GnRH mRNA does not change in response to
pheromones even when measured 45 min after exposure using the
highly sensitive technique, RNAse protection assay to measure GnRH

mRNA, arguing against pheromone-induced changes in GnRH mRNA.

Given that regulation of GnRH depends on where cells reside in
the forebrain, it is important to ask how GnRH subpopulations differ
from one another. GnRH neuronal phenotype and afferents vary with
brain region, a phenomenon that has also been demonstrated other
species (Hoffman et al., 1990;Wu et al., 1992;Mitchell et al.,
1999;Prevot et al., 2000). Anatomical investigations in this
dissertation suggest that neuronal input from GnRH and non-GnRH
neurons also varies with brain region. The cPOA GnRH subpopulation
had the highest proportion of GnRH cells with close appositions of
processes coming from cells in the medial amygdala, an area important

for chemosensory processing (Fiber et al., 1993;Fiber et al.,

147

1996;Kollack-Walker et al., 1997;Swann et al., 1997;Romeo et al.,
1998). GnRH neurons in the cPOA also express the immediate early
gene protein product Fos in response to electrical stimulation of the
vomeronasal organ (Meredith et al., 2001). Taken together, these
studies suggest that medial amygdala projections to GnRH neurons in
the cPOA is one possible pathway by which chemosensory infomiation
can reach the neuroendocrine system. Since cells within many
subdivisions of the medial amygdala are rich with steroid receptors
(Wood et al., 1992;Coolen et al., 1998), it is also conceivable that
steroid negative feedback regulation of GnRH might occur through
action upon cells within the medial amygdala that project to GnRH

neurons.

In addition to input from other neuronal phenotypes, GnRH cells
also receive information from GnRH cells. Communication within the
system is one mechanism by which neuronal activity can be
synchronized, an event necessary for relaying a meaningful peptidergic
signal to the pituitary gland (Witkin, 1999). GnRH-GnRH
communication may be occurring by synaptic and non-synaptic contact
between GnRH cells and ﬁbers (Witkin, 1999). Gap junctions have

been identified in the GnRH immortalized cell line, GT-1 cells, and

148

shown to important for synchronized pulsatile activity of these cells
(Witkin, 1999;Terasawa, 2001;Funabashi et al., 2001;Vazquez-
Martinez et al., 2001). In addition, GnRH cell bodies have been shown
to form an intercellular bridge (Witkin, 1999), another mechanism by
which neuronal activity could be synchronized. Since the greatest
proportion of GnRH neurons with close appositions from GnRH
processes was found in the tenia tecta and MS, and several GnRH cell
bodies were found to be closely apposed to one another in these same
cell groups, it is conceivable that synaptic or non-synaptic
communication within tenia tecta and MS GnRH cells allows for
synchronous activity of GnRH release and this drives the pubertal rise

in gonadotropin release.

In summary, the work presented in this dissertation and
elsewhere (Shivers et al., 1983;King et al., 1987;Berriman et al.,
1992;Tang et al., 1992;Ronchi et al., 1992a;Rubin et al., 1994;1'Anson
et al., 1997;Tang et al., 1997) conveys an overall theme about the
GnRH neuronal system. GnRH cells should not simply be considered
together as a single homogeneous entity, but instead as a complex
neuronal system comprised of separate cell groups whose

responsiveness to various stimuli depends on where they reside within

149

the brain. Function and regulation could differ because of variation in
neuronal input to and receptor expression within different populations
of GnRH neurons. In addition, the types of connections between GnRH
neurons of a subpopulation (i.e., synaptic connection, gap junction, or
bridge-like interceullar connections) likely affects how that cell group
responds to internal or external stimuli. Future investigations of these
potential differences in GnRH subpopulations would enhance our
understanding of how cells that are scattered so far apart could
communicate within and across a subpopulation to synchronize activity

and regulated reproductive status.

150

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