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BRAIN REGIONAL ANALYSIS OF LUTEINIZING HORMONE-RELEASING
HORMONE (LHRH) PRODUCING NEURONS IN THE MALE EUROPEAN FERRET
(Mustela putoriusfuro) DURING PUBERTY

By

Yu Ping Tang

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Neuroscience Program and Department of Psychology

1996

ABSTRACT

BRAIN REGIONAL ANALYSIS OF LUTEINIZING HORMONE-RELEASING
HORMONE (LHRH) PRODUCING NEURONS IN THE MALE EUROPEAN FERRET
(Mustela putoriusfuro) DURING PUBERTY
By

Yu Ping Tang

Activation of the hypothalamic neurons that synthesize and secrete the peptide
hormone LHRH is the proximal event that leads to the onset of puberty. Puberty in male
ferrets is paradoxically associated with a decrease in the number of LHRH-immunopositive
neurons in the arcuate nucleus. This phenomenon was further studied by (1) determining
whether the reduction in LHRH immunoreactivity is primarily within LHRH neurons that
project to the circumventricular organs (mainly the median eminence), or within LHRH
neurons that project elsewhere in the central nervous system; (2) investigating whether the
number of arcuate LHRH neurons is influenced by castration and treatment with
testosterone; and (3) determining whether the decrease in peptide immunoreactivity in
arcuate LHRH neurons is the result of a decrease in LHRH synthesis, as assessed by
intracellular steady state levels of LHRH mRN A. It was found that over 90% of LHRH
neurons in the brain regions examined (preoptic area, retrochiasmatic area of
hypothalamus, arcuate nucleus and median eminence) project to the median eminence or
other circumventricular organs, and that the pubertal decrease in LHRH immunoreactive
cells involves LHRH neurons projecting to the median eminence. Long-term, but not
short-term, castration of prepubertal males resulted in reduced numbers of LHRH-
immunopositive neurons, and testosterone treatment restored LHRH cell number. The
number of arcuate neurons expressing LHRH mRNA increased with pubertal maturation.
Taken together, these results indicate that the pubertal reduction in the number of LHRH

immunoreactive cells is the result of a reduction in cell body stores of LHRH to levels that

are undetectable by immunocytochemistry, and that this reduction in cell body stores is
secondary to a change in the coupling between synthesis and the increased rate of release of

LHRH during puberty.

In memory of my mother,

Li-Chu Weung

iv

ACKNOWLEDGMENTS

I would like to thank my committee members, Drs. Peter Cobbett, Lauren Harris,
Antonio Nunez and Cheryl Sisk. Especially to Dr. Cheryl Sisk, thank you for giving me
the chance to explore science and to become a neuroscientist. I appreciate your
understanding and support through all these years.

Thanks also to Jane Venier, Leslie Meek, Mike Kashon, Kris Krajnak, Kevin
Sinchak. Russ Romeo, Alan Elliott, and Colleen Novak, for their wonderful friendship and
generous assistance over these years.

Finally, thanks to my family for all the love and understanding. I could not do it

without all your support.

TABLE OF CONTENTS

LIST OF TABLES
LIST OF FIGURES
LIST OF ABBREVIATIONS

INTRODUCTION
LHRH Neuronal System in Adults
Pubertal Changes in the LHRH Neuronal System
The Male European Ferret as an Animal Model

GENERAL METHODS
Animals/housing
Castration
Jugular Cannulation
Blood Sampling
Radioimmunoassay of LH and T
Perfusions and Brain Tissue Preparation
Immunocytochemistry of LHRH-containing Neurons
In Situ Hybridization Histochemistry (ISHH)
Microscopic Analysis
Brain regions
Immunocytochemistry
In situ hybridization

EXPERIMENT I. LHRH Neurons Retrogradely Labeled After Peripheral
Administration of Fluoro-Gold in Prepubertal and postpubertal Ferrets
Introduction
Methods
Results
Discussion

EXPERIMENT 11. Effects of Castration and Testosterone on the number of
LHRH-Immunopositive Neurons
Experiment 11A: The effect of short-term castration on LH secretion
and the number of LHRH+ neurons in prepubertal male ferrets
Introduction
Methods
Results
Discussion
Experiment 1le The effect of long—term castration and testosterone
replacement on the number of LHRH+ neurons
Introduction
Methods
Results

vi

viii

ix

22
22
24
25
32

Discussion

EXPERIMENT III. In Situ Hybridization Study of LHRH Neurons During
Puberty

Introduction

Methods

Results

Discussion

GENERAL DISCUSSION
LIST OF REFERENCES

vii

LIST OF TABLES

. Table 1. Mean (iSEM) number of LHRH+ neurons inditified by either LRl antibody or
Hu4H antibody in a 1-in-4 set of 40 um sections within each brain region of postpubertal
male ferrets that were either intact (INT), castrated (CAST) or castrated with TP
replacement (CAST+TP).

Table 2. Mean (iSEM) number of arcuate LHRH+ neurons in a 1-in-4 set of 40 um
sections in pre- and postpubertal intact male ferrets. Data are from Experiment I and IIB
from this dissertation, and from three previous experiments conducted in this laboratory.

Table 3. A summary table of three-way ANOVA for the number of LHRH mRNA
expressing neurons in different age, treatment, and brain region.

Table 4. A summary table of three-way ANOVA for the mean pixels representing silver
grains in neurons in which the number of silver grains was 2 5x background silver grains.

viii

LIST OF FIGURES

Figure 1. Mean (iSEM) number of total and arcuate LHRH+ cell bodies found in every
fourth section through the brain of ferrets at four ages spanning pubertal development.
Asterisks denote values significantly different from lO-wk-old ferrets (p < 0.05).

Figure 2. Saturation curve. Mean (iSEM) pixels per cell at different concentrations of
LHRH cDNA probe.

Figure 3. Line drawings of coronal sections of a representative ferret brain through
forebrain and diencephalon. LHRH—immunopositive neurons within sheded area were
counted for each brain region.

Figure 4. Plasma LH concentrations in individual pre- and postpubertal male ferrets that
received iv injection of either SAL (saline) or NMDA (10 mg/kg) twice daily for 2 days.
An ip injection of 2.5 mg/kg F6 was administered 10 min prior to each SAL or NMDA
injection. Closed circles and open circles represent plasma LH concentrations in blood
samples from day l and day 2, respectively.

Figure 5. Mean (iSEM) LH response in each age and treatment group. The LH response
to SAL (saline) or NMDA injection was calculated as the difference in plasma LH
concentration between the sample just prior to and that just after the injection. Brackets
indicate groups that are significantly different from each other (p < 0.05). Asterisk
indicates significant difference between pre- and postpubertal N MDA groups (p < 0.05).

Figure 6. Photomicrograph illustrating a RIT C labeled LHRH-immunopositive
neuron (indicated by arrow) that was also labeled with PC. Bar: 10 mm.

Figure 7. Mean (iSEM) number of LHRH+ neurons in a 1-in-2 set of 40 um sections
collapsed across treatment in each brain region. Brackets indicate groups that are
significantly different from each other (p < 0.05).

Figure 8. Mean (iSEM) number of double-labeled FG-LHRH+ neurons in a 1-in-2 set of
40 um sections collapsed across treatment in each brain region. Brackets indicate groups
that are significantly different from each other (p < 0.05). Asterisks indicate significant
brain regional differences (p < 0.05).

Figure 9. LH secretory profiles of representative individual ferrets from INTACT, CAST-
3D, CAST-1W and CAST-3W groups. LH secretory profiles in CAST-3W were based on
blood samples at 10 minute intervals, instead of 5 min intervals as for INTACT, CAST-3D
and CAST-1W. Asterisks indicate LH pulses that were recognized by PULSAR program.

Figure 10. Mean (iSEM) LH pulse frequency, LH amplitude and T concentrations from

prepubertal ferrets in INTACT, CAST-3D, CAST-1W and CAST-3W groups. Brackets
indicate groups that are significantly different from each other (p < 0.05).

ix

Figure 11. Mean (iSEM) number of LHRH+ neurons in a l-in-3 set of 50 um sections in
INT, CAST-3D, CAST-1W and CAST-3W prepubertal male ferrets.

Figure 12. Mean (iSEM) number of LHRH+ neurons in a 1-in-3 setof 50 um sections in
three brain regions of prepubertal ferrets. Data are collapsed across treatment groups.
Brackets indicate brain regions that are significantly from each other (p < 0.05).

Figure 13. Mean (iSEM) number of LHRH+ neurons in a 1-in-2 set of 40 um sections in
each brain region of postpubertal male ferrets that were eitherintact (INT), castrated
(CAST), or castrated with testosterone replacement (CAST+TP). Treatment was begun at
10 wk of age and animals were sacrificed at 25 wk of age. Cell counts are based on
combined (averaged) data from the two antibodies (LRl and Hu4H). Brackets indicate
groups that are significantly different from each other (p < 0.05).

Figure 14. Mean (iSEM) plasma testosterone concentration (ng/ml) in INTACT, CAST
(castrate) and CAST+TP (castrate with TP replacement)pre- and postpubertal male ferrets.
Brackets indicate groups that are significantly different from each other within each age (p

< 0.05). Asterisks indicate a significant age difference within each treatment group (p <
0.05).

Figure 15. Photomicrograph illustrating LHRH mRNA expressing neurons
(indicated by arrows) in the arcuate nucleus of intact male ferrets. Bar: 100 um.

Figure 16. Mean (iSEM) number of LHRH mRNA expressing neurons in a 1-in-12 set of
20 um sections within individual brain regions from each age and treatment group.
Brackets indicate that groups are significantly different from one another within each age
and brain region (p < 0.05). Asterisk indicates significant age difference within each brain
region and treatment (p < 0.05).

Figure 17. Mean (iSEM) pixels representing silver grains per LHRH mRNA expressing
neuron within individual brain regions from INTACT, CAST (castrate) and CAST+TP
(castrate with TP replacement) pre- and postpubertal male ferrets.

Figure 18. Mean (iSEM) pixels representing silver grains per LHRH mRNA expressing
neuron collapsed across age and brain region in male ferrets within different treatment

groups. Bracket indicates groups that are significantly different from one another (p <
0.05).

Figure 19. Mean (iSEM) pixels representing silver grains per LHRH mRNA expressing
neuron within each brain region collapsed across treatment groups in pre- and postpubertal

male ferrets. Brackets indicate groups that are significantly different from one another (p <
0.05).

LIST OF ABBREVIATIONS

Anatomical Abbreviations
3V-third ventricle
ARC(arc)-arcuate nucleus
ac-anterior commisure
amy-amygdala
hipp-hippocampus
ME(me)-median eminence
oc-optic chiasm
ot-optic tract
POA(poa)-preoptic area
pvn-paraventricular nucleus
RCH(rch)-retrochiasmatic area
vmh-ventromedial nucleus of hypothalamus

General Abbreviations
ANOVA-analysis of variance
CAST-castrate
CNS-central nervous system
FG-fluorogold
INT-intact
ip-intraperitoneally
ISHH-in situ hybridization histochemistry
iv- intravenous
LH-luteinizing hormone
LHRH-luteinizing hormone releasing hormone
NMDA-N-methyl-D-aspartic acid
RIA-radioimmunoassay
SAL-saline
T-testosterone

xi

INTRODUCTION

Luteinizing hormone-releasing hormone (LHRH) is a neural decapeptide that
plays an essential role in the maturation of the reproductive system in mammals (Matsuo,
Baba, Nair, Arimura, & Schally, 1971). The most important function of LHRH is the
regulation of the synthesis and release of gonadotropins from the anterior pituitary gland,
therefore, LHRH is also known as GnRH (gonadotropin releasing hormone; Schally,
Arimura, Baba, Nair, Matsuo, Redding, Debeljuk, & White, 1971). Gonadotropins
regulate steroid hormone synthesis and release from the gonads. All of these
reproductive hormones are released in a pulsatile manner. In sexually immature animals,
gonadotropin secretion is low because pulsatile LHRH release is low. Thus, an important
component of the onset of puberty in mammals is an increase in the frequency and/or
amplitude of LHRH pulses released into the median eminence (MB) of the hypothalamus,
which leads to an increase in the frequency and/or amplitude of luteinizing hormone (LH)
pulses secreted from the anterior pituitary gland (Bourguinon & Franchimont, 1984;
Terasawa, Claypool, Watanabe, & Gore, 1989; Watanabe & Terasawa, 1989; for reviews,

see Foster, 1994; Ojeda & Urbanski, 1994; Plant, 1994).

LHRH Neuronal System in Adults

The neuroanatomical distribution of LHRH cell bodies within the brain has been
extensively studied by immunocytochemistry in many species (reviewed by Silverrnan,
Livne, & Witkin, 1994). In all species studied, LHRH immunopositive (LHRH+)
neurons are diffusely distributed throughout the forebrain in the olfactory bulb, diagonal

band of Broca, medial septum, preoptic area, and hypothalamus. However, species
1

2

differences in LHRH cell body distribution have also been noted. For example, the
majority of LHRH+ cell bodies in carnivores and primates is located caudal to the optic
chiasm in areas such as the retrochiasmatic area of hypothalamus (RCH) and arcuate
nucleus (ARC) (Boissin-Agasse, Alonso, Roch, & Boissin, 1988; Goldsmith & Song,
1987; King & Anthony, 1984; Tang & Sisk, 1992; Witkin, 1986, 1987a). On the other
hand, most LHRH+ neurons in rodents and ungulates are found in more rostral areas such
as the organum vasculosum of the lamina terminalis, preoptic area (POA), and anterior
hypothalamus (Anthony, King, & Stopa, 1984; Glass, 1986; Jennes & Stumpf, 1980;
King, Tobet, Snavely, & Arimura, 1982; Merchenthaler, Gores, Setalo, Petrusz, &
Flerko, 1984; Schwanzel-Fukuda, Garcia, Morrell, & Pfaff, 1987; Shivers, Harlan,
Morrell, & Pfaff, 1983a; Witkin, Paden, & Silverman, 1982; Wray & Hoffman, 1986a &
b). Unlike carnivores and primates, rodents have few, if any, arcuate LHRH neurons. It
is not known whether these species differences in the neuroanatomical organization of
LHRH neurons have functional consequences.

LHRH immunocytochemical studies have shown that intense LHRH+ fiber
staining is present in the median eminence, suggesting that the median eminence may be
a common final pathway for axonal projections from the scattered LHRH+ cell bodies.
Studies using retrograde tract tracing techniques to investigate the projections of LHRH
neurons found that, although LHRH+ cell bodies are widely distributed within the brain,
most of these LHRH+ neurons do in fact project to the median eminence to regulate
gonadotropin synthesis/release from the anterior pituitary (Berglund & Sisk, 1992;
Goldsmith, Thind, Song, Kim, & Boggan, 1990; Jennes & Stumpf, 1986; Silverman,
Jhamandas, & Renaud, 1987; Silverman, Witkin, & Millar, 1990; Witkin, 1990). Across
these tract tracing studies, the proportion of LHRH+ neurons reported to project to the
median eminence ranges from 35 to 90%. This wide range is probably due to several
factors, including methodological differences, different endocrine status of animals at the

time of sacrifice and possible species differences. In addition to the median eminence,

3

LHRH+ fibers are also found in the amygdala, hippocampus, and preoptic area (for
review see Silverman, et al., 1994). In fact, Jennes ( 1987) was able to demonstrate that
LHRH neurons do terminate in the amygdala and the interpeduncular nucleus by
injecting retrograde tract tracers in these two regions. It has been suggested that LHRH
neurons projecting to the amygdala, interpeduncular nucleus, hippocampus, and preoptic
area may be involved in regulation of reproductive behavior or other processes, rather
than regulation of gonadotropin secretion. More recent findings by Jennes (1991)
reported that about 50% of LHRH neurons showed dual projections to the
interpeduncular nucleus and to the ME, suggesting these neurons may simultaneously act
as a neurohormone and neuromodulator. These results, along with the finding of LHRH
receptors in areas including the diagonal band of Broca, interpeduncular nucleus and
medial septum, support the idea that there is heterogeneity among LHRH neurons, and
that at least some of them play as a neuromodulator (Jennes & Conn, 1994; Jennes &
Woolums, 1994).

Ultrastructurally, LHRH cell bodies are characterized by a large round or ovoid
nucleus, and the presence of Golgi complexes, ribosomes and rough or smooth
endoplasmic reticulum (Kozlowski, Chu, Hostetter, & Kerdelhue, 1980). LHRH neurons
are innervated by catecholaminergic, serotoninergic and opiatergic neurons (Chen,
Witkin, & Silverman, 1989a & b; Kiss & Halasz, 1985; Leranth, MacLusky,
Shanabrough, & Naftolin, 1988a & b; Meister, Hokfelt, Tsuruo, Hemmings, Ouimet,
Greengard, & Goldstein, 1988), all of which may play an important role in the regulation
of activity of LHRH neurons (Gallo, 1980; Kalra, Allen, Sahu, Kalra, & Crowley, 1988;
McCann, 1980; Negro-Vilar, Cullar, & Masotto, 1986; Ramirez, Feder, & Sawyer, 1984;
Wuttke, Roosen-Runge, Demling, Stock, & Vijayan, 1982). However, LHRH neurons
receive relatively few neural inputs compared with other types of neurosecretory cells
(Lehman & Silverman, 1988; Lehman, Karsch, Robinson, & Silverman, 1988; Witkin &
Silverman, 1985; Witkin, 1987b). LHRH cell bodies and dendrites are also innervated by

4

LHRH axons in several species, including the guinea pig, rat, baboon, and monkey
(Chen, Witkin, & Silverman, 1989b; Leranth, Segura, Palkovits, MacLusky,
Shanabrough, & Naftolin, 1985b; Marshall & Goldsmith, 1980; Silverman, 1984;
Silverrnan & Witkin, 1985; Thind & Goldsmith, 1988; Witkin et al., 1982; Witkin &
Silverman, 1985). In contrast, synaptic contact between LHRH neurons was not found in
the hamster (under reproductively stimulatory photoperiods) or anestrous ewe (Lehman
& Silverman, 1988; Lehman, et al., 1988), which may either reflect a significant species
difference, or the relatively rare occurrence of LHRH/LHRH contacts in hamsters and
sheep in a certain reproductive status.

In many studies, the number of LHRH+ cells in the brain remains constant over a
wide range of physiological conditions (Urbanski, Doan, & Pierce, 1991; Urbanski,
Doan, Pierce, Fahrenbach, & Collins, 1992; Wray & Hoffman, 1986b). However,
changes in the number of LHRH+ cell bodies as a function of endocrine state have been
reported. King, Hugel, Zahniser, Wooledge, Damassa, & Alexsavich (1987) found a
significant decrease (about 50%) in the number of LHRH+ cells in male rats one day
after castration. However, six days after castration, the number of LHRH+ cells had
increased compared with one day castrated males. By three weeks postcastration, the
number of LHRH+ neurons in castrated male rats rose more than two-fold compared with
intact male rats. These differences in cell body numbers may be caused by changes in the
rate of synthesis and/or release of LHRH. King, et al. (1987) suggested that acute
gonadectomy in male rats causes an abrupt increase in LHRH secretion and an
exhaustion of LHRH stores within the cell bodies, therefore, fewer numbers of LHRH
neurons are detected immediately after castration. However, long term gonadectomized
rats may re-establish an equilibrium between LHRH synthesis and release that could
account for the subsequent increase in the number of LHRH+ neurons. The number of
LHRH+ neurons in young female rats also changes in association with the preovulatory

LH surge. Rubin and King (1994) reported that the number of LHRH+ neurons in the

5

preoptic area increased in young females during late proestrus compared to young
females in early proestrus. Moreover, the number of LHRH+ neurons increases in the
forebrain, especially in the preoptic area, of the female musk shrew (a reflex ovulator)
after exposure to male-related cues (Dellovade & Rissmann, 1994). Data from these two
studies clearly demonstrate that changes in the number of LHRH+ neurons do occur in
female rodents under different hormonal conditions, and suggest that LHRH+ neurons in
the preoptic area might be important for regulation of the LH surge. Overall, studies
from males and females indicate that the detectability of LHRH+ neurons may be
affected by different hormonal milieus.

Although immunocytochemistry has been used widely to examine the LHRH
neuronal system in animals under different physiological conditions, there are some
limitations with this technique. For example, one can not get direct information about
LHRH synthesis and release with immunocytochemistry. In situ hybridization, which
detects the presence of mRNA within cells, has been used to examine LHRH synthesis
under different hormonal conditions. Most studies have been done in rats, and varying
results have been reported. In males, studies have shown that the number of cells labeled
after in situ hybridization histochemisty is similar in intact and in castrated rats (Malik,
Silverman, & Morrell, 1991; Rothfeld, Hejtmancik, Conn, & Pfaff, 1987; Selmanoff,
Shu, Petersen, Barraclough, & Zoller, 1991; Wiemann, Clifton, & Steiner, 1990).
However, results based on the number of silver grains per labeled cell, which is an index
of intracellular levels of LHRH mRNA, are controversial. Castration has been shown to
result in either an increase (Selmanoff, et al., 1991), decrease (Rothfeld, et al., 1987), or
no change (Malik, Silverman, & Morrell, 1991; Wiemann, Clifton, & Steiner 1990) in the
number of silver grains per labeled cell in male rats. In female rats, the number of
labeled cells and the number of silver grains per labeled cell during the estrous cycle have
been reported either to show no change (Malik, Silverman, & Morrell, 1991), or to

increase on proestrus (Park, Gugneja, & Mayo, 1990; Porkka-Heiskanen, Urban, Turek,

6

& Levine, 1994). The number of silver grains per labeled cell in ovariectomized females
has been shown to either be lower (Pfaff, 1986), or higher (Toranzo, Dupont, Simard,
Labrie, Couet, Labrie, & Pelleter, 1989; Zoller, Seeburg, & Young, 1988), than that in
estrogen-treated ovariectomized females. Clearly, steady state levels of LHRH mRN A
are subject to change, but no clear picture has emerged with respect to steroid regulation.
Direct measurement of LHRH release has been more difficult to study because
accessing the ME or pituitary portal system is difficult. However, recent techniques such
as push-pull perfusion, microdialysis and portal sampling have confirmed much of what
was previously inferred from LH measurements. In female rats, changes in LHRH
secretion from the hypothalamus during the estrous cycle have been reported (Levine,
Bauer-Danton, Beseck, Conaghan, Legan, Meredith, Strobl, Urban, Vogelsong, & Wolf,
1991; Levine & Ramirez 1982; Park & Ramirez, 1989). The preovulatory or estrogen-
induced LH surge is accompanied by an increase in LHRH release in sheep (Caraty,
Locatelli, & Martin, 1989; Clark, 1993; Clark & Cummins, 1985; Moenter, Caraty, &
Karsch, 1990) and monkeys (Chongthammakun & Terasawa, 1991; Levine, Norman,
Gliessman, Oyama, Bangsberg, & Spies, 1985; Pau, Berria, Hess, & Spies, 1993; Xia,
Van Vugt, Alston, Luckhaus, & Ferin, 1992). In male rats. a microdialysis study of the
extracellular hypophyseal fluid showed that mean LHRH levels did not change after
castration, however, LHRH pulse frequency did significantly increase following
castration compared with intact males (Levine & Meredith, 1990). Taken together, these

data suggest that different hormone milieus can affect both LHRH synthesis and release.

Pubertal Changes in the LHRH Neuronal System

A developmental increase in LHRH pulsatile secretion is essential for the onset of
puberty. A study of LHRH release from rat hypothalamic explants demonstrated an age-
related increase in both LHRH content and LHRH pulse frequency (Bourguignon &

Franchimont, 1984). Furthermore, in vivo studies in female rats (Sisk, Shah, & Levine,

7

1996), in female sheep (Foster, 1994) and in monkeys (Watanabe & Terasawa, 1989;
Terasawa, etal., 1989) also showed a pubertal increase in pulsatile release of LHRH.

The LHRH neuronal system has been studied immunocytochemically during
puberty in several species. In most species studied (e.g. rats, Syrian hamsters, and
monkeys), there is no change in the total number of LHRH+ neurons during puberty
(Cameron, McNeil, Fraser, Bremner, Clifton, & Steiner, 1985; Takahashi, Ono, Nomura,
& Kowashima, 1988; Urbanski, et al., 1992; Wray & Hoffman, 1986a). However, two
morphological types of LHRH+ neurons are found in the rat brain (Wray & Hoffman,
1986a & b). "Spiny" LHRH+ neurons have cell bodies and dendrites with either thin
protuberances (pedunculated spines) or knob-like protuberances (sessile spines), and can
be morphologically distinguished from "smooth" neurons which are characterized by the
absence of such protuberances. As puberty progresses, the total number of spiny LHRH+
neurons increases while the total number of smooth LHRH+ cell bodies decreases (Wray
& Hoffman, 1986a). At first, spiny LHRH neurons were thought to have more synaptic
contacts compared to smooth LHRH neurons, indicating that puberty might be associated
with increased synaptic inputs to LHRH neurons (Jennes, Stumpf, & Sheedy, 1985).
However, Witkin and Demasio (1990) found no difference in the number of synapses
between smooth and spiny LHRH neurons by using a systematic morphometric method.
So the functional importance of the pubertal increase in the proportion of spiny LHRH
neurons in rats is still not clear.

A different morphological correlate of puberty is present in Djungarian hamsters.
Juvenile Djungarian hamsters either housed in short day lengths, or given afternoon
injections of melatonin to induce short day reproductive responses, demonstrated delayed
puberty. These hamsters had fewer unipolar LHRH neurons compared to animals that
had undergone puberty in long days (Buchanan & Yellon, 1991). In a subsequent paper,
Yellon and Newman (1991) reported that the number of unipolar neurons increased in the

diagonal band of Broca and in the medial preoptic area at the onset of puberty. A

8

significant increase in the number of bipolar LHRH neurons in the diagonal band of
Broca and the lateral preoptic area also occurred, but was not observed until later stages
of puberty. Thus, in the Djungarian hamster, there is a change in both the number and
morphology of LHRH neurons during puberty. Again, the functional difference between
unipolar and bipolar LHRH neurons has not been established.

A recent ultrastructural study showed that LHRH+ neurons in prepubertal male
rats had more Golgi apparatus and secretory vesicles in the cytoplasm compared with
adults (Witkin & Romero, 1995). LHRH+ terminals on LHRH+ soma were also found in
prepubertal animals, but not in the adults. However, LHRH+ neurons in prepubertal
animals were less well innervated overall than those in adults. Based on these data,
Witkin and Romero (1995) suggested that LHRH+ neurons in prepubertal animals are
actively synthesizing LHRH peptide, but lack integration into the neuronal circuitry.
Moreover, the axosomal synaptic contact between LHRH cells in prepubertal animals
suggested that this pattern of innervation might represent a ultrashort loop negative
feedback circuit (i.e., LHRH inhibition of LHRH synthesis/release) that may be operative
primarily during the period prior to puberty. One study reported that hypothalamic
explants from peri- or postpubertal rats recover from inhibition of LHRH release by an
exogenous LHRH agonist within 35 minutes, whereas explants from prepubertal rats
have a refractory period of over 50 minutes (Bourguignon, Gerard, & Franchimont,
1990). These data suggest that the neural LHRH pulse generating mechanism is more
responsive to this type of inhibitory autofeedback in prepubertal rats than in postpubertal
rats.

There is indirect evidence for an increase in LHRH synthesis during puberty. In
the adult male monkey, the total cross-sectional areas of both LHRH+ perikarya and
cytoplasm are larger than in the juvenile male monkey (Cameron, et al., 1985). In
addition, Takahashi, et a1. (1988) reported that LHRH content in the mid-hypothalamic

area increased in the male rat during puberty. However, despite these indications of

enhanced LHRH synthesis during puberty, Wiemann, Clifton, & Steiner (1989) reported
no increase in LHRH messenger RNA (mRNA) in rats during puberty, in terms of the
level of LHRH mRNA per cell. Despite this, it would seem logical that an increase in
LHRH synthesis would occur during puberty, since LHRH secretion increases. Recently,
Gore, Baum, & Roberts (1995) examined LHRH mRNA and primary transcript levels (an
index of transcription of the proLHRH gene) in male mice during puberty. They found
that LHRH primary transcript levels increased more than 100 fold from postnatal day 5
(P5) to P10 then stayed unchanged from P10 to P 60. Since the stability of LHRH
primary transcript is very low, an increase in LHRH primary transcript might be a
necessary first step for an increase in LHRH mRNA levels. The levels of LHRH mRNA
increased from P5 to P30, and the greatest increase was observed between P25 to P30,
which is just prior to first ovulation. Since much of the increase in LHRH mRNA levels
occurred after primary transcript levels reached a steady—state level (P10), the authors
suggested that the peripubertal increase in LHRH mRNA levels might be due to a post-
transcriptional mechanism such as mRNA stability. The increase in LHRH mRN A
stability would increase mRNA stores and might be important for rapid protein synthesis

at the onset of puberty when the demand of LHRH release is enhanced,

The Male European Ferret as an Animal Model

This laboratory has used the male European ferret (Mustela putoriusfuro) as an
animal model for understanding the neural mechanisms of puberty. The European ferret
is a seasonally breeding species. Under natural photoperiods, the breeding season takes
place in the spring and early summer, when daylengths are increasing (Allanson, 1932;
Bissonette, 1932; Neal, Murphy, Moger & Oliphant, 1977). Young male ferrets born in
early summer undergo puberty around 18 to 20 weeks of age, and their first breeding
season corresponds to the time of annual reproductive recrudescence that occurs in older

adults. In the laboratory, the maturation of the reproductive system in male ferrets can be

10

regulated by changes in environmental photoperiod. The onset of puberty occurs
spontaneously at about 18 wk of age in male ferrets raised under short day lengths (8 hr
light, 16 hr dark), as indicated by marked and rapid testicular growth and by an increase
in LH pulse frequency. However, the onset of puberty can be induced at earlier ages by a
transition from short to long day lengths (18 hr light, 6 hr dark; Sisk, 1990). As in other
male mammals, LH pulse frequency increases during puberty in the ferret (Sisk, 1987).
Since pituitary responsiveness to LHRH is not diminished in prepubertal ferrets
compared to postpubertal ferrets (Berglund & Sisk, 1990), the infrequent secretion of LH
in prepubertal ferrets is assumed to be the result of insufficient LHRH release from the
hypothalamus. The pubertal increase in LH pulse frequency in the ferret is due solely to
a developmental decline in hypothalamic responsiveness to steroid negative feedback on
gonadotropin secretion, since in the absence of gonadal steroids, the pattern of episodic
LH secretion in prepubertal ferrets is comparable to that of gonadectomized adult ferrets.
There is no further increase in the pattern of episodic LH secretion during puberty,
therefore, there is no evidence for a steroid-independent increase in gonadotropin
secretion at the time of puberty (Ryan, Robinson, Tritt, & Zeleznik, 1988; Sisk, 1987).
Gonadal steroids thus either directly or indirectly inhibit LHRH synthesis and/or release
into the median eminence in prepubertal ferrets, and a change in the interaction between
steroid target cells and LHRH neurons must be critical for the onset of puberty in the
ferret.

In an earlier immunocytochemical study, we tested the hypothesis that the
pubertal decrease in responsiveness to steroid negative feedback results in an increase in
the number of neurons that contain LHRH during puberty in the male ferret. Contrary to
our prediction, we found a 50% reduction in the number of arcuate (ARC) LHRH+
neurons in peri- and postpubertal ferrets (20 and 25 weeks old) compared to 10 week old
prepubertal ferrets (Figure l; Tang & Sisk, 1992). Treatment with colchicine, which

inhibits axonal transport, did not reveal additional numbers of LHRH+ neurons in the

ll

arcuate nucleus of postpubertal males, suggesting that the pubertal decrease in arcuate
LHRH+ cell bodies might not be due to a depletion of cell body stores of LHRH resulting
from the pubertal increase in release of peptide (Figure 1).

Since the arcuate nucleus was the only region in which a pubertal change in the
number of LHRH+ cell bodies occurred, it was assumed that these LHRH+ neurons in
ARC play an important role in the onset of puberty in male ferrets. The significant
reduction in the number of ARC LHRH+ neurons, which was first observed at 20 wk of
age, was temporally correlated with the pubertal increase in gonadal size, which also
begins around 20 wk of age. This suggests that the peripubertal disappearance of LHRH
immunoreactivity from approximately half of the ARC LHRH+ neurons may be
functionally related to the enhanced LHRH/LH release that necessarily precedes gonadal
enlargement. If so, a reduction in the number of LHRH+ neurons must be reconciled

with an increase in LHRH secretion.

12

 

240 A. Brain
220 * .coptgl,
200i ucoc cme

180-
1601
140.
120‘
100*
80‘
60‘
40-
20

 

 

 

 

 

 

 

B. Arcuate

Mean (i SEM) Number of LHRH-immunopositive Cell Bodies

 

 

 

     

20
AGE (wk)

Figure 1. Mean (iSEM) number of total (A) and arcuate (B) LHRH+
cell bodies found in every fourth section through the brain of male ferrets
at four ages spanning pubertal development. Asterisks denote values
significantly different from 10-wk-old male ferrets (p < 0.05).

The purpose of this set of experiments is to investigate further the mechanisms of
the pubertal reduction in LHRH immunopositive cell bodies in the arcuate nucleus.
Specifically, they are intended to (1) determine whether the pubertal reduction in ARC
LHRH+ neurons is primarily a reduction in LHRH+ neurons that project to the
circumventricular organs (mainly the median eminence) or is a reduction in LHRH+

neurons that project elsewhere in the central nervous system, (2) investigate whether a

13

change in the number of arcuate LHRH+ neurons is associated with enhanced LH
secretion induced by castration, and (3) determine whether the decrease in peptide
immunoreactivity in arcuate LHRH neurons is the result of a decrease in LHRH synthesis

as assessed by steady state levels of LHRH mRNA.

GENERAL METHODS

Animals/housing

Weanling (7-wk-old) male ferrets were purchased either from Marshall Farms
(North Rose, NY) or the Michigan State University Mink Farm (E. Lansing, MI) and
housed in stainless steel cages (51 x 60 x 38 cm) with Purina ferret Chow (Ralston
Purina, St. Louis, MO) and water available at all times. Ferrets were housed in
temperature-controlled (23 i 1 °C) and light-controlled colony rooms. The lightzdark
(LzD) cycle was either 8L: 16D (short days) or 18L:6D (long days). All protocols were
approved by the MSU All University Committee on Animal Use and Care.

Castration
Testes were removed via a midscrotal incision from ferrets anesthetized with

methoxyflurane anesthesia (Metofane; Pitman-Moore; Washington Crossing, NJ).

Jugular Cannulation

Medical grade Silastic tubing (no. 602-155; id, 0.6 mm; od, 1.19 mm; Dow
Corning, Midland, MI) was cut into 60 cm lengths, flushed with a 2%
tridodecylmethylammonium chloride-heparin complex in toluene (Polysciences,
Warrington, PA) for l min, and then air dried at room temperature. A 2-mm cuff of PE
tubing (no. PE200, Clay Adams, Parsippany, NJ) was sealed 14 cm from one end of the
cannula with super glue. The distal end of the cannula passed through a 30 cm long
spring with an adapter on one end. A three cm length of PE tubing (no. PE60) was sealed
with epoxy inside the male port of a three way stopcock (Baxter Healthcare Corporation,
Valencia, CA). The stopcock was seated in the adapter.

Ferrets were fitted with a jugular cannula under Metofane anesthesia. A

polypropylene cup was sutured to the back of the animal just behind the ears. An
14

15

incision was made above the left jugular vein, then a trochar was passed through the
incision and a hole in the base of the sutured plastic cup. The distal end of cannula was
threaded through the trochar, and the trochar was removed. The spring was secured to
the cup by wire. A small incision was made in the jugular vein and the cannula was
inserted into the vein then anchored above and below the cuff. The skin was closed with
stainless steel autoclips. After surgery, each ferret received 0.4 ml penicillin (im,
300,000 IU/ml; Crysticillin-300-AS, Squibb, Princeton, NJ). The ferret was placed in a
polypropylene cage (40 X 50 X 20 cm), and the protective spring was clamped above the
screen cage top by a ringstand mounted on the side. The cannula was flushed twice a day

with heparinized Krebs—Ringer solution.

Blood Sampling

For a single blood sample, ferrets were anesthetized with Metofane and blood (2
ml) was collected via heart puncture. For frequent sampling, blood samples (0.3-0.5 ml)
were withdrawn via the cannula every 5 or 10 min for up to 4 hr. Blood replacement
(0.6-1.0 ml; washed ferret red blood cells suspended in heparinized Krebs Ringer) was
infused after every other blood sample, therefore, the volume of blood replacement is
equal to the amount of blood that removed. Blood samples were placed in heparinized
tubes on ice until centrifugation. Plasma was removed and stored at -20 °C until

radioimmunoassay.

Radioimmunoassay of LH and T
Plasma concentrations of LH were measured for each sample using the following
reagents: anti-ovine LH GDN no. 15 (obtained from Dr. G. N iswender, Colorado State

University, Fort Collins, CO) and purified ovine LH, LER-1056-C2 (supplied by Dr. Leo

Reichert, Jr., The Albany Medical College, Albany, NY), labeled enzymatically with 1251

l6

and repurified before use on an affinity column containing Concanavalin A-Sepharose
(Pharmacia AB, Laboratory Separation Division, Uppsala, Sweden). Sheep anti-rabbit
gamma-globulin (Antibodies, Inc., Davis, CA) was used to precipitate antibody—bound
hormone. Eight standards were prepared from ovine LH (oLH; NIADDK LH 526,
National Pituitary Agency, Baltimore, MD). The frequency, amplitude and duration of
LH pulses were determined by the PULSAR program (Merriam & Wachter, 1982).
Plasma concentrations of testosterone were measured with reagents in the Coat-a-Count

Total Testosterone Kit (Diagnostic Products, Los Angeles, CA).

Perfusions and Brain Tissue Preparation

Ferrets were anesthetized deeply with Equithesin (2.5 ml/kg, ip) and perfused
intracardially with 350 ml heparinized 0.1 M phosphate buffered saline (PBS), followed
by 350 ml of 4% paraformaldehyde in 0.1 M phosphate buffered saline. The brains were
removed from the skull and stored in 20% sucrose in 4% paraformaldehyde for 2-3 days.
Coronal sections (20, 40 or 50 um, depending on experimental design) through rostral
forebrain and diencephalon were cut on a vibratome or cryostat. Brain sections were

stored in a polyethyleneglycol-based cryoprotectant at -20 °C.

Immunocytochemistry of LHRH-containing Neurons
Sections were washed 3x in 0.1 M PBS with 0.2% Triton X-100 (PBS-TX) and

then incubated in 0.3% H202 in 100% methanol for 30 min to remove remaining

aldehydes and reduce endogenous peroxidase activity. Sections were then incubated
sequentially in normal goat serum (Vectastain ABC Kit, Vector Laboratories,
Burlingame, CA; 30 minutes), rabbit anti-LHRH LR-l (obtained from Dr. Robert Benoit,
The Montreal General Hospital Research Institute) at a dilution of 125,000 in PBS-TX

(16-24 hr), secondary antibody (goat anti-rabbit immunoglobulin; Vectastain ABC Kit; 2

17

hr), avidin-biotin-HRP complex (Vectastain ABC Kit; 1 hr) and diaminobenzidene-
glucose oxidase (Sigma, St. Louis, MO; until reaction product appeared). Sections were
washed 3x in PBS-TX between each incubation. All incubations were at room
temperature, except for that with primary antibody, which was at 4 °C. The reagents
from the Vectastain ABC Kit were prepared according to manufacturer’s instructions.
After the chromogen reaction, sections were washed 5x in PBS, mounted onto gelatinized
slides, dried, counterstained with methylene blue, and coverslipped.

The optimal dilution of primary antibody was determined in pilot experiments in
which a range of dilutions (1:5,000-1:25,000) of primary antibody was tested. For
immunostaining controls, some brain sections were incubated in primary antiserum
which had been preabsorbed with synthetic LHRH (Sigma, St. Louis, MO; 1 jig/ml) for
24 hr at 4 °C. Other brain sections were processed in the absence of primary antiserum.
No immunocytochemical staining of cell bodies or fibers was observed after either

control procedure.

In situ Hybridization Histochemistry (ISHH)

Coronal brain sections (20 um) were cut on a cryostat and thaw-mounted onto
poly-L-lysine-coated slides and vacuum desiccated overnight. Slides were stored at -70
°C until ISHH. ISHH was performed using a 48 base synthetic oligonucleotide
complementary to the LHRH coding region (bases 102- 149) of the human cDNA
(Adelman, et al., 1986). This oligomer was synthesized on an Applied Biosystems 3808
DNA synthesizer and purified with a C18 Sep-Pak cartridge (Waters, Milford, MA). The

probe (15 pmole/ul) was 3' end-labeled by incubating for 60 min at 37 °C with [35S]-
dATP (75 pmole; 1,300 Ci/mmole; New England Nuclear, Boston, MA) and terminal

deoxynucleotidyl transferase (25 U; Boehringer Mannheim, Indianapolis, IN) to a

specific activity of about 106 cpm/ul. The histochemical reaction was similar to

18

published methods for detection of LHRH mRNA (Selmanoff, et al., 1991; Wray, Zoller,
& Gainer, 1989; Zoeller, Seeburg, & Young, 1988). Prehybridization treatment of tissue
consisted of warming the sections to room temperature, incubating for 30 min in 0.001%
proteinase K at 37 °C, followed by 0.0025% acetic anhydride in 0.1 M triethanolamine
(pH 8.0). Sections were rinsed briefly in 2X NaCl/Na citrate(SSC), dehydrated through a
series of ethanols, and dried in a vacuum desiccator. A saturating concentration of
LHRH cDNA probe was determined by applying varying amounts of 35S-labeled probe,
ranging from 50,000 to 1,000,000 cpm/slide (Figure 2).

1 000

 

900 -
800 4
700 -

 

600 -
500 -
400 -

Pixels / Cell (Mean i SEM)

300 .
200 -

 

 

100 . r , f
5.0x 104 2.5 x 105 5.0 x105 1.0 x106

 

LHRH oligoprobe (cpm / slide)

Figure 2. Saturation curve. Mean (iSEM) pixels per cell at different concentrations
of 3SS-labeled LHRH oligoprobe applied to tissue sections.

Based on this saturation curve, 500,000 cpm/slide of labeled probe was applied to

each slide in 100 111 of hybridization buffer consisting of 4X SSC, 50% deionized

19

formamide, 10% dextran sulfate, 25 ug/ml yeast transfer RNA, 500 ug/ml salmon testes
DNA, and 1X Denhardt's solution (0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 0.02%
bovine serum albumin). Slides were coverslipped with parafilm, and hybridization
proceeded for 24 h at 37 °C. Coverslips were removed and sections were then rinsed in
4X SSC and de-salted in decreasing concentrations (IX and 0.5X) of SSC containing 1.0
M dithiothreitol (DTT, 0.1%), then followed by 30 min washes in 0.1X SSC containing
1.0 M DTI‘ at 42 °C. The posthybridization wash temperature was based on analysis of a
melting curve, in which posthybridization temperatures ranged from 37 to 75 °C. In the
melting curve analysis, no labeled cells were found in brain sections that were incubated
in temperatures higher than 65 °C. A final wash in 0.1X SSC containing 1.0 M DTT was
done at room temperature. The slides were dehydrated through a series of ethanols and
dipped in emulsion (NTB-3, Kodak) diluted 1:1 with distilled water. After a 3 week
exposure period, slides were developed in Kodak D-19 developer and counterstained with
methylene blue. Preabsorption of the tissue with an unlabeled probe, or preincubation

with 20 ug/ml RNAse A completely abolished labeling.

Microscopic Analysis
Brain regions
LHRH neurons located in four brain regions, POA, RCH, ARC and ME, were
counted and analyzed. The operationally defined area for each brain region is shown in
Figure 3. In a previous study (Tang & Sisk, 1992), it was determined that about 65% of
all LHRH neurons in the ferret brain are located within these regions. The remaining
35% are scattered outside the four defined brain regions, primarily in the lateral

hypothalamus, diagonal band of Broca, and septum.

20

POA

 

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RCH
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ARC and ME

 

 

 

 

 

 

 

 

 

rostral

 

 

 

 

 

 

 

 

Figure 3. Line drawings of coronal sections of a representative ferret brain through forebrain
and diencephalon. LHRH-immunopositive neurons within shaded area were counted for each
brain region.

21

Immunocytochemistry
Brain sections were examined microscopically under brightfield illumination at
100-1000X. All slides were coded and examined by an experimenter blind to
experimental treatment group. A neuron was identified as LHRH+ if the brown
immunocytochemical reaction product was present within the cytoplasm.
In situ hybridization
The density of silver grains over cells was quantified with a Bioquant Image
Analysis System Meg IV (R & M Biometrics, Nashville, TN). Image analysis was
performed using brightfield optics at 1000X magnification by an experimenter blind to
experimental treatment group. A threshold value was set so that the silver grains
overlying the cell were above, and the underlying cell body was below, threshold. The
cell and the overlying silver grains were then circumscribed by the operator. The area of
the cell body and the number of pixels representing silver grains were determined. The
trace circumscribing the cell was then moved to an adjacent area so that a background of
silver grains was obtained. Cells that had greater than 5 times the number of pixels

representing background silver grains were considered labeled.

EXPERIMENT I. LHRH Neurons Retrogradely Labeled After Peripheral

Administration of Fluoro-Gold in Prepubertal and Postpubertal Ferrets

Introduction

LHRH not only regulates gonadotropin secretion from the anterior pituitary gland,
but it also acts as a neuromodulator within the CNS. Its role as a neuromodulator is
inferred from studies that have demonstrated that LHRH neurons project to areas other
than the median eminence, and that LHRH receptors exist within the central nervous
system (for review see Silverman, et al., 1994). LHRH can also alter
electrophysiological activity of neurons in the hypothalamus (Dudey & Moss, 1987; Dyer
& Dybal, 1974; Moss, 1977; Renuad, Martin, & Brezeau, 1975; Rothfeld, Carstens, &
Gross, 1985) and hippocampus (Palovik & Phillips, 1986). In addition, administration of
LHRH into the preoptic area elicits sexual behavior in ovariectomized female rats primed
with a low dose of estrogen (Rothfeld, Carstens, & Gross, 1985).

In our laboratory, the retrograde tracer Fluoro-Gold (FG) was used in combination
with LHRH immunocytochemistry to examine projections of LHRH neurons in castrated
prepubertal male ferrets. In this study (Berglund & Sisk, 1992), FG was administered as
an ip injection, and could be taken up by nerve terminals outside the blood brain barrier.
With this method, then, it is not possible to distinguish LHRH neurons that project to ME
from those that may project to one of the other circumventricular organs, all of which lie
outside the blood brain barrier. In the Berglund and Sisk (1992) study, approximately
60% of LHRH+ neurons were labeled with FG. This result suggested that the majority of
LHRH neurons are neuroendocrine in nature, that is, they project to the ME and regulate
gonadotropin secretion, but that a subset of LHRH neurons are non-neuroendocrine in
nature, that is, they do not project to ME and presumably subserve some function other
than regulation of gonadotropin secretion. On the other hand, the degree to which FG is

taken up by neuron terminals is thought to be dependent at least in part on neuronal
22

23

activity (Silverman, Witkin, Silverman, & Gibson, 1990). Thus, it is possible that a
greater percentage of LHRH neurons do project to ME in prepubertal ferrets, but are not
sufficiently active to capture FG.

As discussed in the general introduction, an earlier study showed that the number
of arcuate LHRH+ neurons in male ferrets decreased by 50% during puberty (Tang &
Sisk, 1992). One possible explanation for the pubertal reduction in number of arcuate
LHRH+ neurons is that a subpopulation of arcuate LHRH+ neurons may undergo a
change in phenotype during puberty, that is, they may cease production of LHRH. But,
why would this happen? At this point, one can only speculate on the answer to this
question. It is possible that a putative change in phenotype in arcuate LHRH neurons is
unrelated to the pubertal changes in LHRH secretion, but is related to some other,
unidentified pubertal process. If so, then perhaps the peripubertal reduction in arcuate
LHRH immunoreactivity involves a population of neurons that do not project to ME.

It is important to establish whether the pubertal reduction in ARC LHRH+ cells is
a reduction in LHRH cells that project to the MB, or a reduction in LHRH cells that are
non-neuroendocrine in nature. Such information may help to interpret the data, for
example, if the peripubertal reduction in LHRH+ neurons involves only non-
neuroendocrine LHRH neurons, such a finding would suggest that the reduction in cell
number is not directly related to control of anterior pituitary hormone secretion.
Therefore, the goal of this experiment was to use FG as a retrograde tracer, and compare
the number of FG-labeled arcuate LHRH neurons in pre- and postpubertal ferrets. If the
pubertal reduction in LHRH+ neurons is due to a decrease only in non-neuroendocrine
LHRH neurons, then the proportion of FG-labeled LHRH+ neurons in the arcuate of
postpubertal ferrets should be higher compared with that in prepubertal ferrets. Because
of the possibility that differences in neuronal activity could influence the number of FG-
labeled LHRH+ neurons in prepubertal and adult males, retrograde labeling of LHRH+

neurons was also examined in groups of animals that were treated with the excitatory

24

amino acid N-methyl-D-aspartic acid (NMDA). Intermittent iv administration of NMDA
in doses ranging from 5-20 mg/kg b.w. elicits intermittent LH release (Plant, Gay,
Marshall, & Arslan, 1989; Urbanski & Ojeda, 1987). It is believed that NMDA delivered
iv does not cross the blood brain barrier and stimulates LHRH release by action on
LHRH terminals in the median eminence (Gay & Plant, 1987). Thus, pharmacological
activation of LHRH neurons by NMDA should result in uptake of PG by all LHRH
neurons projecting outside the blood brain barrier. Therefore, ferrets receiving NMDA
should have more FG labeled LHRH+ neurons than ferrets without NMDA injections, if,
in our previous study (Berglund & Sisk, 1992), some LHRH neurons were insufficiently

active to take up FG.

Methods

Animals and experimental design

Twenty-two weanling (7-wk-old) male ferrets were purchased from Marshall
Farms (North Rose, NY) and housed in a short photoperiod (8 hr light and 16 hr dark).
At 10 wk of age, 12 prepubertal male ferrets were cannulated and 2 days later, blood
samples were collected at 10 min intervals for one hour. Then ferrets received an iv
injection of either 10 mg/kg NMDA (n=6) or saline vehicle (SAL, n=6), and blood
samples were collected for 1 hr, followed by another NMDA or vehicle injection and 1 hr
sampling period. An ip injection of 2.5 mg/kg FG was administered 10 min prior to the
first NMDA or saline vehicle injection. The same procedure was repeated on the next
day, and cannulas were removed after the final sampling. Two weeks after the last
injection of FG, animals were perfused. The procedure for perfusion and brain tissue
preparation were described in general methods. The remaining 10 male ferrets went
through puberty under short photoperiods and were cannulated at 25 wk of age. The
same procedure for blood sample collection and treatments was used, with 5 NMDA

treated males and 5 saline vehicle treated males. Brain sections (40 um) were cut on a

25

cryostat and every other section from each brain was processed for immunocytochemistry
to identify LHRH neurons. The procedure for LHRH immunocytochemistry was the
same as described previously except that tetramethylrhodamine isothiocyanate-labeled
Avidin D (RIT C; 1:500 dilution, Vector Laboratories) was used for the chromogen
reaction instead of DAB. An immunofluorescent tag must be used for compatibility with
the visualization of PG fluorescence. Sections were examined under epifluorescent
microscopy at 400-1000X to identify neurons labeled with RITC (LHRH+ neurons) and
FG (neurons which project outside the blood brain barrier). Plasma LH concentrations
were determined to confirm that NMDA treatment did result in release of LH. In some
animals, only blood samples from the first day were analyzed for plasma LH levels due to
a limited supply of LH RIA primary antibody.
Data analysis

The LH response to NMDA or saline injection was calculated as the difference in
LH concentration between the sample just prior to and that just after the injection. These
values for the four injections were averaged for each individual and group differences
were analyzed by two-way ANOVA (age by treatment). Group differences in the number
of LHRH+ neurons and double labeled FG-LHRH+ neurons were analyzed by three-way
ANOVA (age by treatment by brain region). Scheffe's F-test was used for post hoc

comparisons.

Results
Plasma LH levels in representative individuals from each group are shown in
Figure 4. Significant main effects of age and treatment, and a significant interaction
between age and treatment were found in the LH response to NMDA and saline
injections. Post hoc comparisons indicated that NMDA elicited a significantly greater
LH response than did saline, and that the LH response to NMDA was greater in

prepubertal males than in postpubertal males (p < 0.05, Figure 5).

26

 

. #738-PRE-SAL-Dl o #861-PRE-NMDA-Dl
1.8 ' o #738-PRE~SAL—D2 o #86l-PRE-NMDA-D2

NMD A NMDA

12 -
SAL

10‘

FG FG
0.6 -

0.4“ f); C
02"

0.0

 

0 #868-POSTSAL-Dl O #87l-PmT-NMDA-D1
1.8 " o #868-POST-SAL—D2 0 #87l-PCBT—NMDA-D2

1.6‘

1.4 '1
SAL

12"

Plasma LH Concentration (ng/ml)

NMDA

1.0 ‘
0.8 "
0.6 "

0.4 -

 

0.2"

 

 

 

 

0.0'I'ir'fi'fi'l'lriifi' rfir'r'rrrfiT'r'rfinfiff

O 20 40 60 80 100 120140 160 180 O 20 40 60 80 100 120140 160 180 200

Time (min)

Figure 4. Plasma LH concentrations in individual pre- and postpubertal male ferrets that received iv
injection of either SAL (saline) or NMDA (10 mg/kg) twice daily for 2 days. An ip injection of 2.5
mg/kg FG was administered 10 min prior to each SAL or NMDA injection. Closed circles and open
circles represent plasma LH concentrations in blood samples from day 1 and day 2, respectively.

27

 

 

 

 

 

 

 

1.0
E
e 0-8- A .
i:
.9. at:
E
g 0.6"
2
8
I: 0.4-
.1
«3
E
g 0.2-
0.1
<
O-
SAL NMDA SAL NMDA
PREPUBERTAL POSTPUBERTAL

Figure 5. Mean (iSEM) LH response in each age and treatment group. The LH
response to SAL (saline) or NMDA injection was calculated as the difference in
plasma LH concentration between the sample just prior to and that just after the
injection. Brackets indicate groups that are significantly different from each other
(p < 0.05). Asterisk indicates significant difference between pre- and postpubertal
NMDA groups (p < 0.05).

28

Intense retrograde Fluoro-Gold labeling was observed in cell bodies in many
brain areas, including the supraoptic, periventricular, paraventricular and arcuate nuclei,
all of which contain large populations of neurosecretory cells that project to the ME or
posterior pituitary gland. The circumventricular organs, i.e., the subfomical organ, the
organum vasculosum of the laminae terminalis, and median eminence, also contained a
large number of FG-labeled neurons. Fluorescent granules of the FG-labeled neurons
were seen mainly in the cytoplasm of the soma and proximal dendrites. LHRH
immunoreactivity appeared as bright red fluorescence in cell bodies and fibers. A
LHRH+ neuron that also contained PC is shown in Figure 6.

Three-way ANOVA revealed significant main effects of age (F(l,69) = 11.273, p <
0.05), brain region (F(3,69) = 97.486, p < 0.05), and significant interaction between age
and brain region (F(3,69) = 3.955, p < 0.05) on the number of LHRH+ neurons. Neither a
main effect of treatment nor interaction between treatment and any other variable was
found in this analysis. Since there was no significant main effect of treatment, data from
each treatment group were collapsed for two-way ANOVA of age and brain region. Post
hoc comparisons indicated that prepubertal males had more LHRH+ cells than
postpubertal males, but only in RCH and ARC (both p < 0.05, Figure 7).

Three-way ANOVA found significant main effects of age (F(l,69) = 7.473, p <
0.05) and brain region (F(l,69) = 87.473, p < 0.05) on the number of double labeled FG-
LHRH+ neurons. There was no effect of treatment and no interaction on this measure.
Post hoc comparisons indicated that overall, prepubertal males had significantly more
double-labeled FG-LHRH+ neurons than postpubertal males (p < 0.05, Figure 8). The
number of double-labeled FG—LHRH+ neurons in the ARC was significantly higher than
that in the POA, RCH, and ME (all p < 0.05, Figure 8).

29

 

Figure 6. Photomicrograph illustrating a RITC labeled LHRH—immunopositive
neuron (indicated by arrow) that was also labeled with FG. Bar: 10 um.

30

 

 

 

 

g 100
§ 90 _ PREPUBERTAL
2‘3 I POSTPUBERTAL
+ 80 "'
i3 0 _
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POA RCH ME

Brain Region
Figure 7. Mean (irSEM) number of LHRH+ neurons in a 1-in-2 set of

40 um sections collapsed across treatment in each brain region. Brackets
indicate groups that are significantly different from each other (p < 0.05).

31

90

80 _ PREPUBERTAL
I POSTPUBERTAL

 

   

70-

@Tu

POA RCH ARC ME

 

Mean (iSEM) Number of Double-Labeled
FG-LHRH+ Neurons

 

 

 

 

 

Brain Region

Figure 8. Mean (iSEM) number of double-labeled FG-LHRH+ neurons in

a 1-in-2 set of 40 um sections collapsed across treatment in each brain region.
Brackets indicate groups that are significantly different from each other

(p < 0.05). Asterisks indicate significant brain regional differences (p < 0.05).

32

Discussion

In this study, the proportion of LHRH+ neurons that were retrogradely labeled
with FG ranged between 75 to 100% across all brain regions examined. Thus, a much
higher percentage of FG-LHRH+ neurons was observed in this study compared with that
observed in a previous study from this laboratory, in which only approximately 60% of
LHRH+ neurons in the preoptic area, retrochiasmatic area of hypothalamus, and arcuate
nucleus were labeled with FG (Berglund & Sisk, 1992). The differences in the
proportion of FG-LHRH+ neurons between these two studies may be due to different
methodologies. In the present study, intact males were used instead of castrate males,
and the post FG-injection survival time was longer (14 days vs. 7-9 days). Since
castration removes testosterone negative feedback and presumably causes an increase in
LHRH secretion from the hypothalamus, LHRH neurons are presumably more active in
castrated animals than in intact animals. In fact, Silverman, et al., (1990) reported that
castrated mice showed a slightly higher pr0portion of double-labeled FG-LHRH+
neurons than intact animals when 2 FG injections were administered, as they were in our
studies. Thus, the proportion of double-labeled FG-LHRH+ cells in prepubertal castrated
males would be expected to be higher in the Berglund and Sisk (1992) study than in the
present study, but this expectation was not confirmed. Therefore, it seems that the longer
survival time used in the present study accounts for the higher degree of retrograde
labeling.

Fluorogold does not cross the blood brain barrier, therefore, when administered
intraperitoneally, it can only be taken up by neurons that have access to the vasculature
through terminals within the circumventricular organs. LHRH fibers within the
circumventricular organs, such as organum vasculosum of the lamina terminalis,
subfomical organ and ME, have been found in most mammals (Silverman, Livne, &
Witkin, 1994), including ferrets (Tang & Sisk, 1992). Thus, LHRH terminals within any

of the circumventricular organs could take up FG and would be identified as double

33

labeled FG-LHRH+ neurons in this study. Since I cannot distinguish LHRH+ neurons
projecting to ME from those projecting to other circumventricular organs, I might be
over-estimating the number of LHRH+ neurons that project to ME and regulate
gonadotropin synthesis/release. Despite this problem of interpretation with using
peripheral injections of FG, studies using direct injection of retrograde tracers into ME
have reported percentages of double labeled LHRH+ neurons ranging from 38 to 70%
(Goldsmith, et al., 1990; Merchenthaler, Setalo, Csontos, Petrusz, Flerko, & Negro-Vilar,
1989; Silverman, Jhamandas, & Renaud, 1987), while the range is 35 to 90% with
peripheral injection of retrograde tracers (Berglund & Sisk, 1992; Jennes & Stumpf,
1986; Silverman, et al., 1990). The higher proportion of double-labeled cells with
peripheral retrograde tracer administration might be due to PG uptake by some LHRH
neurons projecting to circumventricular organs other than ME. The role that LHRH
released from terminals within circumventricular organs other than ME might have on
gonadotropin secretion, if any, has not been explored.

The LH response to NMDA was significantly higher in prepubertal males than in
postpubertal males, suggesting that prepubertal male ferrets are more responsive to
NMDA stimulation than postpubertal males. MacDonald and Wilkinson (1991)
demonstrated that NMDA increased LH levels by 3-fold in prepubertal male rats but did
not increase LH levels in postpubertal male rats. Other studies showed that the LH
response to NMDA was greater in sexually inactive adult animals than in sexually active
adults (Lincoln & Wu, 1991; Meredith, Turek, & Levine, 1991). These results are
consistent with our findings and suggest that the age-dependent difference in the LH
response to NMDA may reflect differences either in the releasable stores of LHRH or
LH. Berglund and Sisk (1990) found that the pituitary LH response to exogenous LHRH
is greater in prepubertal male ferrets than in postpubertal males. Therefore, the different
response to NMDA stimulation between pre- and postpubertal male ferrets might be due

to the differential response of the pituitary gland to LHRH. However, studies have

34

shown that the ability of NMDA to increase LH secretion is mediated by a hypothalamic
mechanism rather than by a direct effect on the pituitary (Bourguignon, Gerard, Mathieu,
Simons, & Franchimont, 1989; MacDonald & Wilkinson, 1990; Ondo, Wheeler, & Dom,
1988; Tal, Price, & Olney, 1983). Tal, Price, and Olney (1983) reported that LH release
from incubated pituitary slices (rats or primates) did not change after NMDA
administration. On the other hand, LHRH secretion from rat hypothalamic slices
increased after NMDA administration (Bourguignon, et al., 1989; Ondo, Wheeler, &
Dom, 1988). An in vivo Estudy also showed that pretreatment with a potent LHRH
receptor antagonist abolished the effect of NMDA on LH secretion in rhesus monkeys
(Gay & Plant, 1987) which also indicated that the effect of NMDA on LH secretion is
due to a hypothalamic site of action. Therefore, in the present study, it is possible that the
greater LH response to NMDA in prepubertal males was due in part to a larger release of
LHRH in response to NMDA, possibly because of larger releasable pools of LHRH.
Although NMDA treatment in prepubertal males did result in a significant
increase in LH secretion and presumably a corresponding increase in LHRH release,
NMDA treatment did not increase the pr0portion of FG-LHRH+ in prepubertal males.
Of course, even in saline treated males, the proportion of double-labeled cells exceeded
90% in RCH, ARC and ME, so it might be difficult to produce a noticeable increase as
the 100% ceiling is approached. It may be that LHRH neurons in prepubertal males are
active, but are secreting peptides or neurotransmitters other than LHRH. In the POA,
76.43% of LHRH+ neurons were labeled with FG in prepubertal SAL treated males, and
this proportion increased to 91% in NMDA treated prepubertal males. This finding
strongly suggests that virtually all LHRH neurons in POA of prepubertal males, like
those in other brain regions, indeed do project outside the blood brain barrier, but
compared with LHRH neurons in other brain regions, POA LHRH neurons are relatively
inactive. Therefore, I conclude that over 90% of LHRH neurons in POA, RCH, ARC and

ME project to ME or other circumventricular organs, and infer from this that the pubertal

35

reduction in ARC LHRH+ neurons most likely reflects a decrease in LHRH
immunoreactivity in neurons that project to the ME. This inference is further supported
by the finding that a pubertal reduction in the number of double-labeled FG-LHRH+
neurons was observed in all four brain regions examined (Figure 8). Since LHRH
secretion increases markedly during puberty, it is possible that the pubertal decrease in
immunoreactivity within LHRH neurons that project to the ME might be due to a
depletion of LHRH cell body stores following enhanced release of LHRH.

A significant pubertal reduction in the number of double labeled FG-LHRH+
neurons, but not in the total number of LHRH+ neurons, was found in the POA and the
ME. However, there was a trend toward a pubertal decrease in the number of LHRH+
neurons in both the POA and the ME. A pubertal reduction was not only found in the
number of double labeled FG-LHRH+ neurons in the ARC, (prepubertal vs. postpubertal:
74.83 vs. 53.0), but was also found in the total number of LHRH+ neurons (prepubertal
vs. postpubertal : 81.58 vs. 53.8), which replicated our previous finding (Tang & Sisk,
1992). Again, these results confirmed our earlier conclusion that the pubertal reduction
in the number of ARC LHRH+ neurons is mainly due to a decrease in the number of
LHRH neurons that project to the ME and regulate LH secretion. In addition, a pubertal
decrease in both the number of LHRH+ cells and double labeled FG-LHRH neurons was
also found in RCH in the present study. A significant reduction in LHRH+ cells was not
observed in RCH in the previous study, although there was a trend in that direction not
only for RCH, but also POA as well. One possible explanation for this discrepancy is
that a higher concentration of primary LHRH-antibody was used in the earlier study
(1:10,000 dilution vs 1225,000 dilution in the present study). Thus, if only a small
amount of LHRH were within LHRH neurons in the RCH, these neurons would be more
likely to be detected in the first study. In any case, the pubertal decrease in LHRH

immunoreactivity appears not to be restricted to the arcuate nucleus.

36

A study of LHRH neuronal projections to ME in the male Djungarian hamster
(Buchanan & Yellon, 1993) demonstrated a significant decrease in the proportion of
LHRH neurons labeled with DiI (a retrograde tracer injected directly into the ME) in
postpubertal males compared with prepubertal males. The authors suggested that fewer
double-labeled LHRH cells in postpubertal males might represent retraction of LHRH
autoinhibitory fibers at puberty. The results from the present study are consistent with
this report, as a pubertal decrease in the number of double-labeled LHRH neurons was
found in POA, RCH, ARC and ME. However, in contrast, a pubertal decrease in the
proportion of double-labeled FG-LHRH+ neurons was not observed in the present study
(data not shown). This discrepancy might be due to a species difference, since there was
no pubertal change in the number of LHRH+ neurons in the male Djungarian hamster and
a pubertal decrease in the number of LHRH+ neurons was found in the male ferret, which
could result in a higher proportion of double-labeled FG-LHRH+ neurons
mathematically.

In summary, this study has demonstrated that over 90% of LHRH neurons in the
brain regions examined project to the ME or other circumventricular organs. It also not
only replicated our earlier finding of a pubertal decrease in the number of ARC LHRH+
neurons, but it also demonstrated a similar age-related decrease in LHRH+ neurons in
RCH. Taken together, these results suggest that pubertal reduction in the number of
LHRH+ neurons is the result of a decrease in immunoreactivity within neurons that
project to ME and regulate LH secretion. Thus, it appears that a pubertal change in
phenotype of LHRH neurons is a less likely explanation of the pubertal reduction in
LHRH neurons than is the possibility that intracellular stores of LHRH are undetectable
in some neurons in postpubertal males. The following experiments will further explore

this latter interpretation.

EXPERIMENT II. Effects of Castration and Testosterone on the Number of

LHRH-Immunopositive Neurons

Experiment IIA: The effect of short-term castration on LH secretion and the

number of LHRH+ neurons in prepubertal male ferrets

Introduction

It was previously thought that the pubertal reduction in ARC LHRH+ neurons
was probably not due to the depletion of detectable LHRH cell body stores after
enhanced release of LHRH in postpubertal males, because blocking axonal transport in
postpubertal male ferrets by colchicine treatment prior to sacrifice did not result in an
increase in the number of LHRH+ neurons in the arcuate nucleus compared with
postpubertal males that did not receive colchicine (Figure l; Tang & Sisk, 1992).
However, it is possible that 24 hr colchicine treatment might not be long enough to
elevate LHRH cell body stores to the detectable level by immunocytochemistry. It is also
possible that the dose of colchicine and/or time of sacrifice after colchicine treatment
used in the previous study were not optimal for complete blockade of axonal transport in
LHRH neurons. Thus, the possibility that the pubertal reduction in the number of
LHRH+ neurons is due to depletion of cell body stores cannot be conclusively ruled out.
Furthermore, the probability that this is the explanation of these data is increased by the
finding from Experiment I that virtually all LHRH neurons project to the ME or other
circumventricular organs. One way to examine this possibility further would be to
determine the number of LHRH+ neurons in prepubertal male ferrets after gonadectomy.
Since gonadectomy, like puberty, is associated with enhanced release of LHRH and LH
(Sisk, 1987), then one would predict a reduction in the number of arcuate LHRH+
neurons after castration, if arcuate LHRH neurons are also actively involved in the

response to the absence of steroid negative feedback after castration.
37

38

As a first test of whether the reduction in arcuate LHRH+ neurons during puberty
is due to enhanced release of LHRH that depletes intracellular stores of LHRH, the
number of arcuate LHRH+ neurons was examined in prepubertal male ferrets that were

gonadectomized in order to remove gonadal steroid negative feedback and to increase

LHRH secretion.

Methods

Animals and experimental design

Twenty four lO-wk-old prepubertal male ferrets housed in short days were
randomly assigned to one of four groups 1) gonad intact (INT, n=9), 2) 3-day castrate
(CAST-3D, n=5), 3) l-wk castrate (CAST-1W, n=5), and 4) 3-wk castrate (CAST-3W,
n=5). On day l (10 wk of age), the 15 ferrets assigned to the castrate groups were
castrated. Five castrated and 3 intact ferrets were cannulated on day 2, and on day 4 (3
days post castration), blood samples were taken from these ferrets at 5 minute intervals
for 4 hours. On day 5, another five castrated ferrets and 3 intact ferrets were cannulated,
and 2 days later (1 wk post-castration), blood samples were collected as before. The
remaining 5 castrated ferrets and remaining 3 intact ferrets were cannulated 19 days post
castration, and blood samples were taken 2 days later (3 wk post-castration). Ferrets in
these last 2 groups were 13 wk of age at the time of blood sampling, and therefore intact
males were still in the prepubertal state at this time. Following the blood sampling
period, all ferrets were anesthetized and perfused as described in the General Methods.
In this experiment, brain sections were cut with a vibratome at 50 pm. Every third brain
section from the preoptic area through the mammillary bodies was processed for LHRH
immunocytochemistry. All sections were mounted onto gelatinized slides, dried,
counterstained with methylene blue, coverslipped and examined microscopically under

brightfield illumination at 100-400X.

39

Blood samples were assayed for LH concentrations. However, at the time the
initial LH RIA was run, the primary antibody for the LH assay was no longer available,
and I had only a limited amount of this antibody on hand. Therefore, LH in samples
obtained from the CAST-3W group and the 3 INT ferrets sampled at the same time was
not measured. Recently. I obtained a small amount of primary antibody GDN15 from Dr.
M. Baum. Therefore I was able to measure LH in every other plasma sample (i.e., at 10
min intervals) in the CAST-3W ferrets and the contemporary cohort of intact ferrets.
After the LH RIA, the remaining plasma in every other blood sample from each ferret
was pooled together for the T RIA. The minimum detectable concentration for the two
LH and the T RIAs were 0.19, 0.15 and 0.15 ng/ml, respectively. The intraassay CVs for
LH were 12.32 and 19.35%. The interassay CV for LH was 15.84%. The assay CV for
T was 13.1%.

Data analysis

LH pulse frequency and amplitude were analyzed by the PULSAR computer
program. The G values in the PULSAR program, which define the number of assay CV5
over a smoothed baseline that one. two, three, four, or five elevated points must be to
qualify as a pulse, were G(1)=2.0, G(2)=1.2, G(3)=1.2, G(4)=1.0, and G(5)=1.0.

To compare data collected from the 3 cohorts of intact males sampled at the 3
times corresponding to CAST-3D, CAST-1W, and CAST-3W, the number of LHRH+
neurons in POA, RCH, and ARC, and mean T concentrations in these intact ferrets, were
first compared by separate one-way AN OVAs. Parameters of LH secretion (pulse
frequency and pulse amplitude) were similarly compared in the three groups of INT
ferrets. This analysis showed no significant differences among the three cohorts of INT
ferrets on any of these hormonal or neuroanatomcal measures. Therefore, data from all
intact prepubertal males were combined into a single group for statistical comparisons
with the castrated groups. The number of LHRH+ neurons in POA, RCH and ARC was

analyzed by two-way ANOVA (treatment by brain region). Group differences in LH

40

pulse frequency, LH pulse amplitude, and mean T concentrations were all analyzed by
separate one-way ANOVAs. Scheffe's F-test was used for post hoc comparisons.

Differences were considered statistically significant when p < 0.05.

Results

Figure 9 shows the pulsatile pattern of LH release in representative individuals
from the INT, CAST-3D, CAST-1W and CAST-3W groups. ANOVA revealed a
significant effect of treatment on LH pulse frequency (F1210) = 17.407, p < 0.05) and LH
pulse amplitude (F(2.20) = 6.645, p < 0.05). Scheffe's post hoc comparisons indicated that
mean LH pulse frequency of INT ferrets was significantly lower than that of CAST-3D (p
< 0.05), and of CAST-1W ferrets (p < 0.05). The CAST-3W group also had a lower LH
pulse frequency than did the CAST-3D group (p < 0.05) (Figure 10). Mean LH pulse
amplitude was significantly higher in CAST-1W and CAST-3W males compared to INT
males (both p < 0.05) (Figure 10). There was no effect of treatment on plasma T
concentrations, even though testosterone was largely undetectable in castrated ferrets
(Figure 10).

Despite the fact that LH pulse frequency and amplitude were increased after
castration, neither a main effect of treatment nor an interaction between brain region and
treatment on the number of LHRH+ neurons was observed (both p > 0.05, Figure l 1).
There was a significant main effect of brain region on the number of LHRH+ neurons
(F(3,54) = 180.02, p < 0.05). Post hoc comparisons showed that ARC had significantly
more LHRH+ neurons than POA and RCH. The number of LHRH+ neurons in RCH

was also higher than in POA (Figure 12).

Discussion
In this experiment, castration resulted in the expected increase in LH pulse

frequency in the CAST-3D and CAST-1W groups, and in LH pulse amplitude in the

41

 

 

 

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T1ME(min)

Figure 9. LH secretory profiles of representative individual ferrets from INT ACT , CAST-3D, CAST-1W
and CAST-3W groups. LH secretory profiles in CAST-3W were based on blood samples at 10 minute
intervals, instead of 5 min intervals as for INTACT, CAST-3D and CAST-1W. Asterisks indicate LH pulses
that were recognized by PULSAR program.

 

42

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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INTACT CAST-3D CAST- 1 W CAST-3W
Treatment

Figure 10. Mean (i'SEM) LH pulse frequency. LH amplitude and T concentrations from
prepubertal ferrets in INTACT, CAST-3D, CAST-1W and CAST-3W groups. Brackets
indicate groups that are significantly different from each other (p < 0.05).

43

 

 

 

 

 

 

 

 

 

 

 

 

 

 

g 80

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5 70 - INT

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Figure 11. Mean (iSEM) number of LHRH+ neurons in a 1-in-3 set of 50 um
sections in INT, CAST-3D, CAST-1W and CAST-3W prepubertal male ferrets.

70

 

 

60-

50-

40-

30-

20-

 

 

Mean (iSEM) Number of LHRH+ Neurons

 

POA RCH ARC

Figure 12. Mean (iSEM) number of LHRH+ neurons in a l-in-3 set
of 50 um sections in three brain regions of prepubertal ferrets. Data are
collapsed across treatment groups. Brackets indicate brain regions that
are significantly from each other (p < 0.05).

45

CAST-1W and CAST-3W groups. LH pulse frequency in CAST-3W male ferrets was
about 0.45 pulses/hr, which was not significantly different from INT males (0.17
pulses/hr). However, pulse amplitude did significantly increase in CAST-3W group
compared to INT group (0.6 vs. 0.07 ng/ml, respectively). In a previous study from our
laboratory (Sisk, 1987), LH pulse frequency was about 0.7 pulses/hr in prepubertal male
ferrets 3 weeks after castration. Therefore, the LH pulse frequency in the CAST-3W
group of this study is somewhat lower than previously found. Since LH pulse frequency
in CAST-3W was analyzed based on a 10 min sampling interval, rather than the 5 min
interval used for analysis in the other groups in this study and in the earlier study, it is
possible that some pulses could have been missed in the CAST-3W group.

No difference in the number of LHRH+ neurons between intact and castrated
ferrets was observed in any brain region for up to 3 weeks after castration. Therefore, an
increase in LH secretion does not immediately result in a decrease in the number of
LHRH+ neurons in the arcuate nucleus. However, depletion of cell body stores of LHRH
after castration may require a longer period of time than 3 weeks. It is likely that 3 weeks
of relatively frequent LH release following castration is not equivalent to the prolonged
increase in LH secretion experienced over the course of puberty, which in the Tang and
Sisk (1992) study would have been ongoing for approximately 10 weeks prior to the time
that the postpubertal animals were sacrificed (25 wk of age). In additon, coupling
between synthesis and release could be different under conditions of castration and
pubertal maturation.

The present results in the prepubertal male ferrets are different from what has
been reported in the adult rat. King, et al., (1987) found that one day after castration of
adult male rats, plasma LH increased and the number of LHRH+ neurons decreased. But
at six days after castration, the number of LHRH+ neurons returned to the intact level as
plasma LH continued to rise. By three weeks post castration, the number of LHRH+

neurons was significantly increased above intact levels. Their findings indicated there

46

are rapid changes in LHRH immunoreactivity after removal of negative feedback, and
also suggested that the rate of release might be greater than the rate of synthesis in the
castrate condition, at least immediately after castration. On the other hand, Witkin (1989)
reported that while no change in the number of LHRH+ neurons was observed in adult
male rats 1 day after castration, a significant decrease in the number of LHRH+ neurons
was found in rats 4 weeks after castration as compared to 1 day castrates. It is not clear
whether the discrepancy in the changes of the number of LHRH+ neurons after castration
between the King, et al. (1987) and Witkin (1989) studies is due to the length after
castration or to different methodologies. Since I did not examine the number of LHRH+
neurons immediately after castration, it is not known whether a rapid and transient
decrease in LHRH immunoreactivity might have occurred in the prepubertal male ferret,
or whether changes in LHRH cell body number would occur later than three weeks after

castration.

Experiment IIB: The effect of long-term castration and testosterone replacement on

the number of LHRH+ neurons

Introduction

In the previous experiment, there was no change in the number of LHRH+
neurons for up to three weeks postcastration. However, 3 weeks postcastration in
prepubertal male may not be long enough to deplete cell body LHRH stores and result in
a reduction in arcuate LHRH neurons. This experiment examined the effect of long-term
castration and testosterone replacement on the number of LHRH+ cells in adult male
ferrets that were castrated prepubertally. If the pubertal reduction in the number of
arcuate LHRH+ neurons is due to the enhanced release of LHRH at puberty and

subsequent depletion of cell body stores of LHRH, it is predicted that a decrease in the

47

number of arcuate LHRH+ neurons would still occur, and perhaps be even more
pronounced, in adult males that were castrated prepubertally. Two different anti-LHRH
antibodies, LRl, which recognizes both the preprohorrnone for LHRH and the mature
decapeptide, and Hu4H, which recognizes only the mature decapeptide, were used in this
experiment. The rationale for using these two antibodies was to allow a comparison of

the proportion and the distribution of cells containing only the mature LHRH decapeptide

with those containing the preprohormone in the presence and absence of testosterone.

Methods

Animals and experimental design
Fifteen prepubertal male ferrets housed in short days remained either gonadally

intact (INT, n=5), or were castrated at 10 wk of age. Beginning at the time of castration
all ferrets received a daily subcutaneous injection of either oil (INT and CAST, n=5) or
testosterone propionate (CAST+TP, 5 mg/kg in sesame oil, n=5) for the remainder of the
experiment. All ferrets were transferred to long days (LD; 18 hr light and 6 hr dark) at 12
wk of age to induce puberty in the INT group. All ferrets were sacrificed and perfused at
25 wk of age. Blood samples (via heart puncture) were obtained at the time of sacrifice,
and were assayed for plasma T concentrations. Each brain was cut into 4 sets at 40 um
thickness from the diagonal band of Broca through the mammillary bodies. Half of the
brain sections (sets 1 and 3) was processed with LRl antibody (1225,000 dilution), and
the other half (sets 2 and 4) was processed with Hu4H antibody (1:2,000 dilution) for
LHRH immunocytochemistry. After the chromogen reaction, brain sections from set 1
(using LR 1) and set 2 (using Hu4H) were mounted onto gelatinized slides, dried,
counterstained with methylene blue, and coverslipped. Brain sections from set 3 (using

LR l ) and set 4 (using Hu4H) were mounted onto gelatinized slides, dried, and

coverslipped without counterstaining.

48

Data analysis
The effect of treatment on the number of LHRH+ neurons in POA, RCH, ARC,

and ME were analyzed by two-way ANOVA (treatment by antibody). Scheffe's F-test
was used for post hoc comparisons. This analysis revealed no effect of antibody, and no
interaction between treatment and antibody on the total number of LHRH+ neurons in

POA, RCH, ARC, and ME (Table 1). Therefore, the cell counts based on the two

antibodies were combined (averaged) for a two-way ANOVA analysis (treatment by
brain region).

Results
The minimum detectable concentration for the testosterone RIA was 0.14 ng/ml,
and the assay CV was 1 1.8%. The mean plasma concentrations of testosterone were
significantly different among the three treatment groups at the time of sacrifice (25 wk of
age; F(2,11) = 22.381, p < 0.05). Post hoc comparisons revealed that mean plasma
testosterone concentration in the CAST+TP group (26.9 i 3.9 ng/ml) was significantly

higher than that the INT and CAST groups (6.55 i 2.67 ng/ml and 0.14 i- 0.0 ng/ml,

respectively).
Two-way AN OVA showed significant main effects of treatment (F(2, 40) =

15.91 1, p < 0.05), brain region (F(3,40) = 62.018, p < 0.05), and a significant interaction
between treatment and brain region (F(6,40) = 3.317, p < 0.05) on the number of LHRH+

neurons. Scheffe's post hoc comparisons indicated that the numbers of LHRH+ neurons

in RCH, ARC and ME, but not in POA, were significantly lower in the CAST group

compared with the CAST+TP group (all p < 0.05, Figure 13).

Discussion

When cell counts are normalized to a 1-in—4 set of sections, the number of arcuate

LHRH+ neurons in postpubertal intact males in this experiment was comparable to that

49

Table 1. Mean (iSEM) number of LHRH+ neurons identified by either LRl antibody or
Hu4H antibody in a 1-in-4 set of 40 um sections within each brain region of

postpubertal male ferrets that were either intact (INT), castrated (CAST) or castrated
with TP replacement (CAST+TP).

 

INT CAST CAST+TP
LRl Hu4H LRl Hu4H LRl Hu4H

POA 15.75 :1: 5.81 10.50 i- 4.92 7.25 i 2.63 7.50 i 1.04 14.60 i 4.27 12.80 i 5.22
RCH 26.75 i 2.46 22.00 i 1.68 13.75 i 2.78 17.75 :L' 3.35 31.00 i 5.21 30.80 i 3.69
ARC 52.00 i 6.01 63.75 i 14.06 30.25 i 7.43 32.00 i 7.82 68.20 i 8.86 80.40 i 6.15
MB 5.00 i 1.47 7.50 i 3.10 2.50 i: 0.29 1.25 i 0.25 10.60 i 2.25 12.00 i 2.10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

50

 

90

80 - INT ' '
[I CAST
7° ‘ CAST+TP

6O -
50 a
40 -
30
20
10

 

 

l

 

1

 

V
.4

 

 

 

 

s\\\\\\\\\\\s—~

 

 

 

 

 

 

 

 

 

 

Mean (iSEM) Number of LHRH+ Neurons

 

POA ARC ' I} ME

Figure 13. Mean (iSEM) number of LHRH+ neurons in a 1-in-2 set of 40 um
sections in each brain region of postpubertal male ferrets that were either

intact (INT), castrated (CAST), or castrated with testosterone replacement
(CAST+TP). Treatment was begun at 10 wk of age and animals were sacrificed
at 25 wk of age. Cell counts are based on combined (averaged) data from the
two antibodies (LRl and Hu4H). Brackets indicate groups that are significantly
different from each other (p < 0.05).

51
observed in postpubertal intact males in three previous experiments (Table 2).
Therefore, I infer that a pubertal reduction in the number of arcuate LHRH+ neurons

occurred in intact males in the present experiment. That is, bad there been a group of

males sacrificed at the time of the beginning of treatment (10 wk of age), arcuate LHRH+

neuron number would have been approximately twice that of the intact adults in this

‘1

study (see numbers for ARC LHRH+ neurons in 4 previous studies, Table 2).

In adults that had been castrated for 15 weeks, the number of arcuate LHRH+

neurons was approximately half that of aged matched intact adults (although not
statistically different). Earlier studies from this laboratory have shown that in male
ferrets, there is a pubertal increase in LH pulse frequency, presumably due to increased
LHRH release (Sisk, 1987). Moreover, LH pulse frequency can be further increased in
adults by castration (Sisk & Desjardins,'1986). Taken together, the results from
Experiment HA and IIB provide indirect evidence that the pubertal reduction in the
number of arcuate LHRH+ neurons seen in intact males may be due to depletion of
LHRH stores that is secondary to the pubertal increase in LHRH secretion. This
conclusion is based on the observation that there appears to be a loose negative
correlation between the number of arcuate LHRH+ neurons and demand for LHRH
secretion. Intact and short-term castrated prepubertal male ferrets have a similar number
of arcuate LHRH+ neurons, which is greater than that of adult intact males, and adult
intact males have more arcuate LHRH+ neurons than long-term castrated adults.
Conversely, the demand for LHRH release is greatest in adult long term castrates and is
lowest in prepubertal intact males. This idea is further supported by the data from
CAST+TP group in Experiment IIB, in which the number of arcuate LHRH+ neurons
was significantly greater than that of long-term castrates. The daily testosterone
injections clearly resulted in high plasma concentrations of testosterone, which
undoubtedly inhibited LHRH and LH secretion, and which could prevent the depletion of

cell body stores of LHRH normally seen in long-term castrates. Thus, the data from this

 

 

1‘

 

 

 

52

Table 2. Mean (iSEM) number of arcuate LHRH+ neurons in a 1-in-4 set of 40 um
sections in pre- and postpubertal intact male ferrets. Data are from Experiment I and IIB
from this dissertation, and from three previous experiments conducted in this laboratory.

 

 

 

 

 

 

 

 

 

 

EXPERIMENT PREPUBERTAL POSTPUBERTAL
Experiment IIB - 28.9 i 3.71
ExperimentI 47.3 i 2.19 27.1 i 2.82
Peptides (1992) 13:241-247 58.0 i 5.81 27.5 i 4.56
Puberty 7.1 (unpublished) 42.9 i- 3.25 26.0 i 4.02
J. Neuroendo. (1992) 42743-749 54.7 i 5.20 -

 

53

set of experiments suggest that the length of time and degree to which LHRH release is
enhanced, the higher the probability of a decrease in the number of ARC LHRH+
neurons.

In an earlier study, colchicine treatment 24 hr prior to sacrifice did not result in
additional numbers of LHRH+ neurons in the arcuate nucleus of postpubertal males,

which led to an initial interpretation that the pubertal decrease in arcuate LHRH+ cell
bodies might not be due to depletion of cell body stores of LHRH (Tang & Sisk, 1992).
However, if in the postpubertal male, the rate of synthesis of LHRH in arcuate neurons is
no greater than the rate of release, it is still possible that LHRH would not be detectable

in the soma within 24 hr of blocked axonal transport.
In RCH and ME, there was a greater number of LHRH+ neurons in the CAST+TP

compared with the CAST group. Previously, a pubertal reduction in LHRH+ neurons
was observed only in the arcuate nucleus, although there was also a trend toward reduced
numbers of LHRH+ cells in ME (Tang & Sisk, 1992). In addition, results from
Experiment I of this dissertation also showed an age-related decrease in the number of
LHRH+ neurons in both ARC and RCH, and a trend toward reduced numbers of LHRH+
cells in POA and ME. Perhaps LHRH neurons in the arcuate nucleus respond first to the
increased demand for LHRH secretion during puberty and therefore are more likely to be
subject to depletion of LHRH cell body stores. With additional demands for hormonal

output with long-term castration, perhaps other populations of LHRH neurons are

susceptible to depletion of cell body stores.
The number of LHRH+ neurons that reacted with either LRl or Hu4H antibody

was similar within the brain regions examined. This finding suggests that the processing
of LHRH preprohorrnone to mature decapeptide occurs in the soma. In the rat,
Silverman, Witkin, and Millar (1990) examined where in the neuron LHRH processing
occurs, and reported that the processing and cleavage of the LHRH precursor molecule

began in the cell soma. Ronnekleiv, Adelman, Weber, Herbert, and Kelly (1987) also

1

 

54

demonstrated that the processing of LHRH precursor to the biologically active LHRH
decapeptide in the rhesus macaque and the baboon primarily occurs in the cell soma. In
contrast, King and Anthony (1983) failed to find mature LHRH decapeptide within
perikarya and suggested that the processing of LHRH occurred in neuronal fibers and
terminals. The discrepancy may be due to different fixation procedures in those studies.
In the present study, no effect of antibody and no interaction between treatment
and antibody was observed on the number of LHRH+ neurons in any brain region
examined. Thus, the LHRH+ cells in all three treatment groups contained mature LHRH
decapeptide, and T may affect the LHRH neuronal system at the level of release rather
than at the level of preprohorrnone processing in adult intact and longterrn castrated male

ferrets.

EXPERIMENT III. In Situ Hybridization Study of LHRH Neurons During
Puberty

Introduction

Data from Experiment II suggest indirectly that the pubertal decrease in LHRH
immunoreactive cells may reflect depletion of LHRH cell body stores as a consequence
of increased LHRH release during puberty. A direct test of this hypothesis would require
in vivo measurement of LHRH release, which is not presently feasible to do in this animal
model. It is possible to directly test the alternative hypothesis, however, and that is that
the decrease in LHRH+ cells reflects a decrease in the number of LHRH synthesizing
cells. Therefore, in situ hybridization histochemistry (ISHH) was used in this study to
identify cells that express LHRH mRNA in pre- and postpubertal male ferrets. A second
goal of this experiment was to see whether testosterone affects LHRH mRNA expression.
Studies of the effects of testosterone on LHRH mRNA expression in adult male rats have
yielded conflicting results (Gruenewald & Matsumoto, 1991; Malik, Silverman, &
Morrell, 1991; Rothfeld, et al., 1987; Selmanoff, et al., 1991; Toranzo, et al., 1989;
Wiemann, Clifton, & Steiner, 1990; Wray, Zoller, Gainer, 1989; see also general
introduction). It is still not clear whether testosterone negative feedback on LHRH is at
the level of synthesis, release or both. Since prepubertal male ferrets are more sensitive
to testosterone negative feedback on LHRH/LH secretion than postpubertal males, I
wanted to test whether there is a differential response of LHRH mRN A expressing

neurons to testosterone in pre- and postpubertal male ferrets.

Methods
Animals and experimental design
Twenty eight gonadally intact prepubertal male ferrets (7—wk-old) were housed in

short days (8 hr light, 16 hr dark). Half of these ferrets were randomly assigned to be
55

56

treated prepubertally and the other half were assigned to be treated postpubertally. Of the
14 prepubertal subjects, nine ferrets were castrated at 8 wk of age. Two weeks after
castration, five of these castrated ferrets received a testosterone propionate (TP, 5 mg/kg
sc) injection once a day for two weeks. The remaining four castrated ferrets received
control injections of oil. At the end of the two week period of treatment (12 wk of age),
these ferrets and the five remaining intact ferrets were perfused. Blood samples were
collected by heart puncture before perfusion. The fourteen postpubertal subjects were
transferred from short days to long days (18 hr light and 6 hr dark) at 12 wk of age to
induce and synchronize the onset of pubertal maturation. Under these photoperiodic
conditions testis width is maximal within 8 wk of the photoperiod transition. As with the
prepubertal groups, nine postpubertal ferrets were castrated at 20 wk of age and allowed
to recover for 2 weeks. Five of them then received TP injections and another four
received 011 injections once a day for two weeks. These ferrets and the remaining five
intact ferrets were sacrificed at 24 wk of age. Blood samples and brain tissue were
collected as for prepubertal ferrets. Tissue sections (20 pm, a l-in-12 set) from all
treatment groups were processed together for ISHH for LHRH mRN A (see general
methods). Plasma T concentrations were determined by RIA.
Data analysis

Plasma concentration of T was analyzed by two-way ANOVA (age by treatment).
For brain regional analysis, the number of LHRH mRNA expressing cells and the
average pixels per cell in POA, RCH, ARC and ME were analyzed by three-way
ANOVA (age by treatment by brain region). Scheffe's F-test was used for post hoc

comparisons.

57

Results

Plasma Concentrations of Testosterone

The minimum detectable concentration of T was 0.1 1 ng/ml and the assay CV
was 12.96%. There was a significant interaction between age and treatment on plasma
concentrations iof testosterone (F(2,21) = 5.187, p < 0.05; Figure 14). Post hoc
comparisons indicated that postpubertal males had significantly higher concentrations of
T than prepubertal males in both the INT and CAST+TP groups (both p < 0.05). The
difference between pre- and postpubertal CAST+TP was unexpected, since the dose of
TP was adjusted to body weight.
Brain Regional Analysis

A. Number of LHRH mRNA expressing neurons

Photomicrographs of LHRH mRNA expressing neurons are shown in Figure 15.
Brain regional analysis (three-way ANOVA, age by treatment by brain region) of the
number of LHRH mRN A expressing neurons (based on cell counts in a one-in-12 sets of
sections) is shown in Table 3. There were significant main effects of age, treatment, and
brain region. Significant interactions between age and treatment, between age and brain
region, and between treatment and brain region were also observed. Furthermore, there
was a significant three-way interaction among age, treatment, and brain region. Because
of the significant 3-way interaction, any two independent variables would produce a
different pattern of results at different levels of the third variable. Therefore, the data
were further analyzed by examining the age and treatment interaction within each brain
region (Figure 16).

In both POA and ME, there were no significant main effects of either age or
treatment, and no interaction between age and treatment.

In RCH, there were significant main effects of age (p < 0.05) and treatment (p <
0.05), as well as a significant interaction between age and treatment (p < 0.05). Post hoc

comparisons showed that treatment affected the number of LHRH mRNA expressing

58

 

 

 

 

 

 

   

 

 

 

 

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INTACT CAST CAST+TP INTACT CAST CAST+TP

PREPUBERTAL POSTPUBERTAL

Figure 14. Mean (iSEM) plasma testosterone concentration (ng/ml) in
INTACT, CAST (castrate) and CAST+TP (castrate with TP replacement)
pre- and postpubertal male ferrets. Brackets indicate groups that are
significantly different from each other within each age (p < 0.05). Asterisks
indicate a significant age difference within each treatment group (p < 0.05).

59

 

Figure 15. Photomicrograph illustrating LHRH mRNA expressing neurons
(indicated by arrows) in the arcuate nucleus of intact male ferrets. Bar: 100 um.

60

Table 3. A summary table of three-way ANOVA for the number of LHRH mRNA
expressing neurons in different age, treatment, and brain region.

DF Sum of Squares Mean Square F-Value P-Value

 

 

 

 

 

 

 

age 1 291.054 291.054 14.403 .0003
treatment 2 260.246 130.123 6.439 .0025
age * treatment 2 174.936 87.468 4.329 .0162
brain region 3 4887.036 1629.012 80.615 <.0001
age * brain region 3 236.256 78.752 3.897 .01 16
treatment * brain region 6 277.256 46.209 2.287 .0428
age * treatment * brain region 6 431.643 71.940 3.560 .0034

 

 

 

 

 

 

Residual 85 1717.617 20.207

 

 

61

 

4o
POA RCH
35 - g] lNTACl'

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30 - I CAST+TP

 

 

 

 

 

 

 

 

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35-
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PREPUBERTAL POSTPUBERTAL PREPUBERT AL POSTPUBERTAL

 

Mean (iSEM) Number of LHRH mRNA Expressing Neurons
g

 

 

 

 

 

 

 

   

 

 

 

 

 

 

 

 

AGE

Figure 16. Mean (iSEM) number of LHRH mRNA expressing neurons in a 1-in-12 set of 20 um sections
within individual brain regions from each age and treatment group. Brackets indicate that groups are
significantly different from one another within each age and brain region (p < 0.05). Asterisk indicates
significant age difference within each brain region and treatment (p < 0.05).

62

neurons only in postpubertal males, where CAST+TP males had more LHRH mRN A
expressing neurons than INT males (p < 0.05).

In ARC, there were significant main effects of age and treatment, and a
significant interaction between age and treatment (all p < 0.05). Post hoc comparisons
showed that significant treatment effects on the number of LHRH mRN A expressing
neurons were found only in prepubertal male ferrets (p < 0.05), and not in postpubertal
males. In prepubertal males, CAST males had a significantly higher number of LHRH
mRN A expressing neurons than INT males (p < 0.05).

B. Labeling intensity (silver grains/cell) of LHRH mRNA expressing neurons

Table 4 and Figure 17 show the results of the brain regional analysis (three-way
ANOVA, age by treatment by brain region) on the mean pixels per labeled cell. This
analysis is based on quantification of pixels that represented silver grains over all cells
that were classified as labeled in the previous analysis (i.e., number of silver grains over
the cell 2 5x background). There were significant main effects of age (F(l,79) = 12.029, p
< 0.05), treatment (F(2,79) = 4.996, p < 0.05), and brain region (F(3,79) = 17.389, p <
0.05), as well as a significant interaction between age and brain region (F(6.79) = 3.98, p <
0.05). Neither an interaction between age and treatment nor an interaction between brain
region and treatment were observed. Post hoc comparisons to analyze the main effect of
treatment indicated that CAST males had significantly higher mean pixels per cell than
C AST+TP males (p < 0.05, Figure 18).

Since there was no significant three-way interaction among age, treatment, and
brain region, the data from each treatment group were collapsed for analyzing the two-
way interaction between age and brain region (Figure 19). Post hoc comparisons showed
th at the mean pixels per labeled cell were greater in postpubertal males compared with
prepubertal males, but only in POA and ARC (both p < 0.05). In postpubertal males
only, the mean number of pixels per cell in POA was significantly higher than in any

other brain region (all p < 0.05).

63

Table 4. A summary table of three-way ANOVA for mean pixels representing silver
grains in neurons in which the number of silver grains was 2 5x background silver
grams.

DF Sum of Squares Mean Square F-Value P-Value

 

age 565181.113 565181.113 12.029 .0009

 

treatment 469447.817 234723.908 4.996 .0091

 

age * treatment 207602.829 103801.415 2.209 .1165

 

brain region 2451062243 817020.748 17.389 <.0001

 

561015.599 187005.200 3.980 .0107
treatment * brain region 534676.940 891 12.823 1.897 .0917
age * treatment * brain region 538247.788 89707.965 1.909 .0895

age * brain region

 

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Residual 79 371 1821.190 46985.078

 

 

64

 

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1600 q
1400 -
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1000 -
800 .,
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1600 ,
1400 .
1200 .
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Mean (iSEM) Pixels Per LHRH mRNA Expressrng Nneuron
G
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POSTPUBERTAL PREPUBERTAL

AGE

POSTPUBERTAL

Figure 17. Mean (iSEM) pixels representing silver grains per LHRH mRNA expressing neuron within
individual brain regions from INTACT, CAST (castrate) and CAST+TP (castrate with TP replacement)

pre- and postpubertal male ferrets.

65

 

 

 

 

 

 

 

 

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INTACT CAST CAST+TP
Treatment

Figure 18. Mean (+SEM) pixels representing silver grains per LHRH mRNA
expressing neuron collapsed across age and brain region in male ferrets within
different treatment groups. Bracket indicates groups that are significantly
different from one another (p < 0.05).

<—lfiflfil --~--- I «IIIA- .1-1.1.|ull I“..IVI < \ I I

 

 

 

 

 

66

 

       

 

 

 

 

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POA RCH ARC ME

Brain Region

Figure 19. Mean (iSEM) pixels representing silver grains per LHRH mRNA
expressing neuron within each brain region collapsed across treatment groups
in pre- and postpubertal male ferrets. Brackets indicate groups that are
significantly different from one another (p < 0.05).

67

Discussion

In contrast to our previous immunocytochemical studies, which showed that
postpubertal male ferrets have significantly fewer ARC LHRH-immunopositive neurons
compared to prepubertal males, the results from this study show that postpubertal males
have more LHRH mRNA expressing neurons in ARC than prepubertal males. Therefore,
the pubertal decrease in the number of ARC LHRH+ cells does not reflect a decrease in
the number of ARC LHRH synthesizing cells. In fact, I estimate that the number of ARC
LHRH mRNA expressing neurons in postpubertal males is roughly comparable to the
number of ARC LHRH+ neurons in prepubertal males (280 vs. 240, respectively). The
numbers of LHRH mRN A expressing neurons in Experiment IH and LHRH+ neurons in
Tang and Sisk (1992) were based on counts of a 1-in-12 set and a l-in-4 set of sections,
respectively, in each brain region. Therefore, these numbers were then multiplied either
by 12 or by 4 respectively for the estimated total numbers of LHRH mRNA expressing
neurons (approximately 80 in prepubertal and 280 in postpubertal males) and LHRH+
neurons (approximately 240 in prepubertal and 120 in postpubertal males) in the whole
arcuate nucleus. If it is assumed then, that the number of arcuate neurons that are of the
LHRH phenotype is the same in pre- and postpubertal males, then the pubertal decrease
in ARC LHRH immunoreactivity is in all likelihood due to depletion of LHRH cell body
stores. I cannot rule out the possibility that LHRH message is not being translated into
protein, but this seems a less likely interpretation of these data.

In prepubertal male ferrets, approximately 240 LHRH+ neurons are found in the
whole arcuate nucleus, while only 80 neurons are found to express LHRH mRNA. A
likely explanation for these numbers is that synthesized LHRH peptide is stored in LHRH
cell bodies but because the demand for secretion of LHRH is low in prepubertal males,

many neurons are not actively expressing LHRH mRNA.

68

The increase in the number of LHRH mRNA expressing neurons in postpubertal
males was found in the arcuate nucleus only. One interpretation of this brain region
specificity is that arcuate LHRH neurons might be important for testosterone negative
feedback regulation of gonadotropin secretion. Therefore, these arcuate LHRH neurons
in prepubertal males might be very sensitive to testosterone negative feedback on LHRH
synthesis and/or release. During puberty, these arcuate LHRH neurons become less
sensitive to testosterone negative feedback, resulting in more LHRH neurons
synthesizing and/or releasing LHRH in this region, but not in others. This might explain
why more arcuate LHRH mRNA expressing neurons were observed in postpubertal
males than in prepubertal males.

While other studies have shown no pubertal changes in either the number of
LHRH cells labeled after in situ hybridization histochemistry in the monkey (Vician,
Adams, Clifton, & Steiner, 1991) or in the intensity of labeling per cell in male rats
(W iemann, Clifton, & Steiner, 1989) and monkeys (Vician, et al., 1991), our data showed
that a pubertal increase occurs not only in the number of ARC LHRH mRNA expressing
neurons in male ferrets, but also in the intensity of labeling per cell in both POA and
ARC. The pubertal increase in LHRH mRNA within individual neurons in ARC might
be due to the decrease in sensitivity to testosterone negative feedback that results in
enhanced release of LHRH. Since there are very few LHRH mRNA expressing neurons
in POA, the biological significance of the pubertal changes in labeling intensity in POA
is not clear. However, LHRH synaptic inputs onto LHRH somata in POA were found
only in prepubertal male rats, and not in adult male rats (Witkin & Romero, 1995). If this
finding is also true for male ferrets, then it is possible that LHRH neurons might exert
ultrashort100p inhibitory feedback on POA LHRH neurons in prepubertal males. After
pubertal maturation, the ultrashort100p inhibitory feedback in POA might be removed due
to the lack of synaptic contacts between LHRH neurons, which would result in an

increase of LHRH synthesis within the individual LHRH neurons in POA. Since

69

castration in prepubertal males did not change in either the number of LHRH mRN A
expressing neurons or the labeling intensity per cell in POA, it is unlikely that the
pubertal increase in labeling intensity in POA is due to the decrease in sensitivity to
testosterone negative feedback.

In prepubertal male ferrets, castration significantly increased the number of
LHRH mRN A expressing neurons in ARC only, and this number was comparable to the
number of ARC LHRH mRN A expressing neurons in postpubertal intact males. On the
other hand, castration had no effect on the number of LHRH mRNA expressing neurons
in any brain region of postpubertal males. These data suggest that arcuate LHRH
neurons in prepubertal male ferrets are more sensitive to testosterone negative feedback
than those in postpubertal males, which further supports the idea that the pubertal
increase in the number of ARC LHRH mRNA expressing neurons is related to the
pubertal decrease in sensitivity to testosterone negative feedback.

The lack of an effect of castration on the number of labeled cells in postpubertal
male ferrets is consistent with other studies in adult male rats, which also found no effect
of castration on the number of LHRH mRNA expressing cells (Malik, Silverman, &
Morrell, 1991; Rothfeld, et al., 1987; Selmanoff, et al., 1991; Wiemann, Clifton, &
Steiner, 1990). The effect of castration on LHRH mRNA expressing neurons in
prepubertal animals of other species has not been reported. In terms of the effect of
castration on labeling intensity per cell, studies have reported either no change (Malik,
Silverman, & Morrell, 1991; Wiemann, Clifton, & Steiner, 1990), a decrease (Rothfeld,
et al., 1987), or an increase (Selmanoff, et al., 1991; Toranzo, et al., 1989) compared to
intact animals. The present study showed no effect of castration on labeling intensity per
cell in either pre- or postpubertal males. However, castration with TP replacement
significantly decreased labeling intensity compared with oil-treated castrates, indicating

that at least pharmacological treatment with T can reduce LHRH gene expression.

7O

Toranzo, et a1. (1989) also reported that the labeling intensity was higher in castrated
male rats than in castrated male rats with either E or DHT replacement.

The effects of castration and TP replacement on the number of LHRH mRN A
expressing neurons in the RCH of postpubertal males is puzzling. The number of labeled
cells in RCH was greatest in the CAST+TP group, and the number of labeled cells in the
CAST group was intermediate between that of intacts and TP treated castrates. Plasma
testosterone levels in postpubertal CAST+TP males were significantly higher than that in
postpubertal intact males. High concentrations of T in CAST+TP male ferrets might
pharmacologically result in an inhibition of inhibitory inputs to RCH LHRH+ neurons,
resulting in an increase in LHRH synthesis. Brain regional differences in steroid
metabolizing enzymes or glial/neuronal relationships might also explain the anomalous
results in RCH. If aromatase activity is unusually high in RCH, it could result in a
decrease in local concentrations of testosterone that are insufficient to exert negative
feedback on LHRH synthesis. It is also possible that testosterone might affect the
interaction between glial cells and LHRH neurons in RCH in an unusual way, which
could result in a testosterone—related increase in LHRH synthesis in this brain region.

In this study, prepubertal male ferrets were housed under short photoperiod while
postpubertal males were housed under long photoperiod after being transferred from
short to long photoperiod at 12 weeks of age. Therefore, photoperiod was a confounding
variable in this experiment, and could influence sensitivity to testosterone, as does age.
In this laboratory, it was previously found that there was no difference in gonadal size
and in the pulsatile release of LH between adult male ferrets that had gone through
puberty either spontaneously (under short daylengths) or after photo-induction
(transferred from short to long daylengths) (Sisk, 1990). Thus, it is likely that had there
been an additional group of adults raised in short days, testosterone would have exerted a
similar effect on LHRH mRNA as it did in the present group of adults transferred to long

days.

71

In contrast to various effects of age and steroid hormone on LHRH mRNA
expressing neurons in POA, RCH, and ARC, neither age nor steroid hormone treatment
had any effect on the number of labeled cells or labeling intensity of LHRH mRNA
expressing neurons in ME. Although LHRH mRNA expressing neurons in ME are most
close to ARC, however, they do not respond to the testosterone treatment in the same
way as ARC LHRH neurons. It is an interesting and puzzling result that remains to be

answered with further studies.

GENERAL DISCUSSION

This set of experiments suggests that the pubertal reduction in the number of
arcuate LHRH+ neurons is due to a depletion of LHRH stores within some LHRH
neurons following enhanced LHRH secretion during puberty. This conclusion is based
on converging lines of evidence from several aspects of the data. First, a decrease in the
number of arcuate LHRH+ neurons was observed not only in adult gonad intact males,
but also in adult males that had been castrated prior to puberty. Furthermore, these long
term adult castrates tended to have even fewer arcuate LHRH+ neurons than intact adults,
suggesting that the greater the demand for LHRH release, the greater the likelihood of a
decrease in immunoreactive cell bodies. Second, using in situ hybridization
histochemistry to identify LHRH synthesizing cells, a pubertal increase was observed in
the number of labeled cells in the arcuate nucleus, which suggests strongly that the
decrease in immunoreactivity is not due to a cessation of LHRH synthesis in a
subpopulation of arcuate LHRH neurons. Third, short term castration of prepubertal
males resulted in an increase in the number of LHRH mRNA expressing cells in the
arcuate that was similar to the number in adult intact males, indicating that the increase in
LHRH release stimulated by removal of steroid negative feedback results in increased
LHRH synthesis. Finally, virtually all LHRH neurons in the arcuate nucleus project to
the MB (or to other structures outside the blood brain barrier), indicating that the vast
majority of these cells do participate in the regulation of gonadotropin secretion from the
anterior pituitary gland. Therefore, the most simple explanation for the pubertal decrease
in LHRH immunopositive cell bodies is that over the course of puberty, cell body stores
of LHRH become undetectable in many LHRH producing cells not only in the arcuate
nucleus, but also in the retrochiasmatic area of hypothalamus and ME (Experiments 1 and

IIB).

72

73

One question raised by these experiments is why the pubertal increase in LHRH
synthesis within ARC would not prevent the putative depletion of LHRH stores that leads
to a decrease in the number of ARC LHRH+ neurons. It is known that, as male ferrets go
through puberty, they become less sensitive to testosterone negative feedback in terms of
enhanced pulsatile release of LH and presumably LHRH (Sisk, 1987). Thus, it is
possible that the rate of LHRH synthesis in postpubertal males simply keeps up with the
rate of LHRH secretion, and that newly synthesized LHRH peptide is transported to the
terminals right away.

Although I tried in Expt. 11a to mimic the pubertal increase in LHRH release in
prepubertal male ferrets by castration, I failed to find any changes in the number of ARC
LHRH+ neurons for up to 3 weeks after castration in prepubertal males. However,
effects on ARC LHRH+ cell numbers were seen 15 wk after castration. As mentioned
earlier, one possible explanation for not seeing a decrease in LHRH+ cell body number
after only 3 weeks is that this might not be a long enough time to render LHRH cell body
stores undetectable. Another possible explanation is that the rate of LHRH synthesis may
be increased acutely after castration, and this might retard the depletion of cell body
LHRH stores. This is supported by evidence from Experiment III which showed that
castration in prepubertal male ferrets significantly increased the number of ARC LHRH
mRNA expressing neurons.

It is possible that removal of testosterone negative feedback in prepubertal male
ferrets by castration is not equivalent to the pubertal decrease in sensitivity to testosterone
negative feedback in terms of how it might affect the number of LHRH+ neurons. An
ultrastructural study by Witkin and Romero (1995) found that LHRH neurons in
prepubertal male rats were less innervated than those in adult males, suggesting that the
integration of LHRH neurons into the circuitry is not complete in prepubertal males. If
this is also true for male ferrets, then any change in LHRH synthesis and release might be

regulated by different mechanisms under short term castration and pubertal maturation.

74

In addition, Witkin (1989) examined the effect of gonadectomy on the synaptology of
LHRH neurons in adult male rats and found no change in the density of synaptic inputs to
LHRH neurons 4 weeks postcastration. In an aging study (Witkin, 1987), an age-related
increase in the density of synapses on LHRH neurons was found in old adult male rats,
which presumably had lower serum testosterone levels (Ghanadian, Lewis, & Chisholm,
1975). Taken together, these findings suggest that developmental or physiological
changes in steroid hormone levels may have different effects on the LHRH neuronal
system than do the less physiological changes in steroid hormone levels that accompany
castration.

Ultrastructural studies have demonstrated that LHRH neurons are innervated by
several different cell phenotypes, including those that contain B-endorphin (Chen, Witkin,
Silverman, 1989b; Leranth, et al., 1988b), GABA (y-aminobutyric acid; Leranth,
MacLusky, Sakamoto, Shanabrough, & Naftolin, 1985a), serotonin (Kiss & Halasz,
1985), catecholamines (Chen, Witkin, & Silverman, 1989a; Watanabe & Nakai, 1987;
Leranth, et al., 1988a), vasopressin (Thind, Boggan, & Goldsmith, 1991), CRH
(corticotropin releasing hormone; MacLuskey, Naftolin, & Leranth, 1988), or glutamate
(Goldsmith, Thind, & Perera, 1992). Among these neurosubstances, GABA, the
catecholamines, and B-endorphin have been shown to have effects on LH secretion
(Barraclough & Wise, 1982; Kalra & Kalra, 1984; Lamberts, Vijayan, Graf, Mansky, &
Wuttke, 1983). In addition, estrogen receptors are present in neurons that contain
GABA, the catecholamines, and B-endorphin (Flugge, Oretel, & Wuttke, 1986;
Jirikowski, Merchenthaler, Rieger, & Stumpf, 1986; Morrell, McGinty, Pfaff, 1985; Sar,
1984). Moreover, galanin, a neuropeptide, can stimulate LHRH release from nerve
terminals of the median eminence in vitro (Lopez & Negro-Vilar, 1990), and galanin-
containing cells also contain androgen receptors (Bingaman, Baeckman, Yrcheta, Handa,
& Gray, 1994). Since the vast majority of LHRH cells do not express either estrogen or

androgen receptors (Huang &Harlan, 1993: Lehman & Karsch, 1993; Shivers, Harlan,

75

Morrell, & Pfaff, 1983b; Sullivan, Silverman, Witkin, & Ferin, 1990; Watson, Langub, &
Landis, 1992), it is likely that gonadal steroids affect LHRH synthesis and release
indirectly by acting on steroid-responsive neurons afferent to the LHRH neurons.

Recently, the role of glial cells in the regulation of the LHRH neuronal system has
received increased attention. Glial cells have not only been found to ensheathe LHRH
neurons (Witkin, Ferin, Popilskis, & Silverman, 1991; Witkin, O’Sullivan, & Ferin,
1995), but changes in glial morphology have also been found under different steroid
conditions (Brawer, Schipper, & Robaire, 1983; Garcia-Segura, Luquin, Parducz, &
Naftolin, 1994; McQueen, Wright, Arbuthnott, & Fink, 1990; Tranque, Suarez, Olmos,
Fernandez, & Garcia-Segura, 1987; Witkin, et al., 1991). Ovariectomy of female rhesus
monkeys resulted in a significant increase in the apposition of glial processes to LHRH
perikarya] membranes in POA and RCH compared with that in intact females (Witkin, et
al., 1991). Moreover, glial cells also express estrogen receptor (Langub & Watson, 1992;
Santagati, Melcangi, Celotti, Martini, & Maggi, 1994). Therefore, gonadal steroids may
regulate LHRH synthesis and release through glial cells by altering the apposition
between glial processes and LHRH perikarya] membrane. As mentioned earlier, gonadal
steroids may regulate the amount of LHRH reaching the anterior pituitary by acting on
glial elements in terms of changing the accessibility of LHRH terminals to the basal
lamina in MB. This hypothesis is supported by the finding that the distance of LHRH
terminals in ME from the basal lamina of the brain decreased after castration (King &
Letoumeau, 1994). It is possible that brain region differences in terms of the LHRH
neuronal response to testosterone in the male ferret may be due to differences in
LHRH/glial interactions in different brain regions.

Using multiple three-dimensional computerized reconstructions of LHRH-‘
immunopostive neurons in the female rat brain during proestrus and estrus, it was
demonstrated that the distribution of LHRH neurons was most widespread during

proestrus and was most restricted during estrus (Hiatt, Brunetta, Seiler, Barney, Selles,

76

Wooledge, & King, 1992). On the basis of this evidence, it has been suggested that
LHRH neurons are organized into heterogeneous subgroups in female rats (Hiatt, et al.,
1992). The subgroups of LHRH neurons reveal a laminar organization, which may
reflect a gradient of activity within the population of cells, from more midline to more
lateral neurons and from more rostral to more caudal neurons. The female ferret is a
reflex ovulator, in which mating induced ovulation is the result of an increase in LH
secretion in response to somatosensory stimulation during mating (Carroll, Erskine,
Doherty, Lundell, & Baum, 1985; Carroll, Erskine, & Baum, 1987). In the female ferret,
the proportion of LHRH-immunoreactive neurons colabeled with c-fos increased after
mating (Lambert, Rubin, & Baum, 1992). This finding suggests that the LHRH neuronal
system in female ferrets may also show a gradient kind of activity. However, there was
no apparent brain regional organization to the activation of LHRH neurons induced by
mating.

In our earlier study (Tang & Sisk, 1992), a pubertal reduction in the number of
LHRH+ neurons was observed only in the arcuate nucleus when a higher concentration
of anti-LHRH antibody was used (1:10,000). However, a pubertal decrease in the
number of LHRH+ neurons was found not only in the arcuate nucleus, but also in
retrochiasmatic area of hypothalamus, in Experiment I, when a lower concentration of
anti-LHRH antibody was used (1:25,000). In addition, a trend toward an age-related
decrease in the number of LHRH+ neurons was observed in the preoptic area. Taken
together, our data suggest that in male ferrets, either during puberty, or in response to
castration, when the demand for LHRH secretion increases, it is most likely that LHRH
neurons within ARC will be the first to respond, followed by LHRH neurons in RCH and
POA. If so, this also suggests a layered-type organization of LHRH neurons within the
male ferret brain that may reflect a caudal to rostral gradient of activity of LHRH

neurons.

77

Further studies are necessary to gain more insight into mechanisms of changes in
the LHRH neuronal system in the male ferrets during puberty. For example, using
microdialysis for direct measurement of LHRH release, examining the changes in glial
and LHRH neuronal apposition during puberty, and investigating the morphological
changes of glial cells under different steroid conditions could all provide important

information.

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