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The Japanese quail: a model for studying

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Article in Experimental Gerontology November 2004
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Experimental Gerontology 39 (2004) 16791693

www.elsevier.com/locate/expgero

Review

The Japanese quail: a model for studying reproductive aging

of hypothalamic systems
Mary Ann Ottingera,*, Mahmoud Abdelnabia, Qichang Lia, Kehong Chena, Nicola Thompsona,
Nobuhiro Haradab, Carla Viglietti-Panzicac, Gian Carlo Panzicac
a

Department of Animal and Avian Sciences, University of Maryland, College Park, MD 20742, USA
Department of Biochemistry, School of Medicine Fujita Health University, Toyoake, Aichi 470-1192, Japan
c
Department of Anatomy, Pharmacology and Forensic Medicine, University of Turin, Torino, Italy
Received 17 May 2004; accepted 14 June 2004
Available online 7 October 2004

Abstract
During aging, the decline of neuroendocrine, endocrine, and behavioral components of reproduction ultimately leads to reproductive
failure. These studies considered both neuroendocrine and behavioral aspects of reproductive aging in Japanese quail, using chronological
age and reproductive status to separate animals into experimental groups. In Study I, age-related changes in the gonadotropin releasing
hormone (GnRH-I) system were investigated and a sharp decrease was observed in GnRH-I concentration in the median eminence of aging
animals of both sexes, whereas preoptic-lateral septal region GnRH-I concentrations declined only in aging males. Immunohistochemistry
confirmed these findings since aging females retained, whereas males lost GnRH-I cells. Functional changes were assessed by in vitro
incubation of parasaggittal hypothalamic slices collected from young and old inactive males and females. Results showed reduced baseline
GnRH-I release and diminished response to norepinephrine (NE). Deteriorating fertility also correlated with decreased male sexual behavior
and loss of aromatase immunoreactive (AROM-ir) neurons in the medial, but not lateral preoptic nucleus (POA). Sexual behavior and
AROM-ir were restored with exogenous testosterone, which was associated with increased cell size in the medial POA. Comparison of cell
size and number of AROM-ir cells showed that aged sexually active males had fewer, larger AROM-ir cells when compared to young males,
suggesting neuroplasticity of specific neural systems and a critical role of estradiol in maintaining reproductive function.
q 2004 Elsevier Inc. All rights reserved.
Keywords: Japanese quail; Age-related reproductive decline; GnRH-I and aging; Neurotransmitters and aging

1. Introduction
In mammals and other vertebrates, aging in healthy
individuals is associated with slowing or diminished
function of physiological, metabolic, reproductive, and
sensory systems leading to impaired function and response.
As normal aging occurs in men, there is a gradual loss of
both endocrine and behavioral components of reproductive
function (Veldhuis, 1997; Ferrini et al., 2001; Lejeune et al.,
2003). In women, the hormonal loss is more precipitous,
resulting in hot flashes, ovarian dysfunction, and other
symptoms of perimenopause (Wise, 1998). In both, there

* Corresponding author. Tel.: C1 301 405 5780; fax: C1 301 314 9059.
E-mail address: maotting@umd.edu (M.A. Ottinger).
0531-5565/$ - see front matter q 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.exger.2004.06.021

may be increasing pathologies during aging, such as

prostate hyperplasia and endometriosis. A wealth of studies
have established fundamental events that occur during the
aging process and the effects of treatments, such as calorie
restriction on individual rates of aging in a variety of animal
models (Weindruch and Walford, 1982; Nelson et al., 1985;
Mobbs et al., 2001; Lane et al., 2002). In order to understand
the complexities of the process of aging, it is useful to have
simpler models for study. Our studies have developed the
Japanese quail as a model to characterize lifetime
reproductive function and the mechanisms involved in
reproductive aging.
The Japanese quail is a well-known model for studying
hypothalamic and limbic circuits involved in the control of
reproductive axis (for a review see Ottinger et al., 1997;
Ottinger, 1998; Panzica et al., 2001). In addition, several

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M.A. Ottinger et al. / Experimental Gerontology 39 (2004) 16791693

studies were dedicated to characterizing lifetime of

reproductive function of the mechanisms involved in
reproductive agining at each level of the reproductive
axis. Therefore, the Japanese quail provides an advantageous model for investigating neuroendocrine and behavioral components of reproduction in the context of aging.
We will summarize our earlier studies and present original
data on neuroendocrine and behavioral components of
reproductive aging.
Our studies have characterized the process of aging in
males and females, beginning with an overview of lifetime
reproduction (Ottinger et al., 1983; Balthazart et al., 1984;
Ottinger and Balthazart, 1986; Ottinger and Bakst, 1995;
Ottinger, 2001). By 18 months of age, pairs show decreased
reproductive function, and reduced fertility. However,
females appear to age more rapidly than males (Ottinger
and Bakst, 1995; Ottinger et al., 1995). We have studied the
hypothalamic-pituitary-gonadal (HPG) axis, to identify the
timing of functional changes at each level during aging. Our
findings have shown that the age-related deterioration in
morphology and function occurs at each level of the HPG
axis (Ottinger et al., 1995, 1997, 2002a,b; Ottinger, 1998).
Aging males have more gonadal abnormalities and tumors,
particularly Sertoli cell tumors in spite of maintained
Leydig cell function and diminished spermatogenesis
(Gorham and Ottinger, 1986). Moreover, both LH and
FSH receptor number sharply decrease with reproductive
aging and testicular regression. Interestingly, exogenous
testosterone resulted in increased LH, but not FSH receptor
number (Ottinger et al., 2002b). Declining hypothalamic
response to gonadal steroids appears to be an early event in
aging in both quail and domestic chickens (Williams and
Sharp 1972; Sharp et al., 1992; Ottinger et al., 1997). In
males, this is accompanied by decreased male sexual
behavior and in females egg production becomes increasingly irregular (Palmer and Bahr1992; Ottinger, 2001).
Further, altered hypothalamic neurotransmitters and neuropeptides appear to be fundamental to the functional changes
inherent in the cascade of events leading to reproductive
failure (Ottinger et al., 1997).
These studies revealed several unique characteristics
and advantages of Japanese quail as a model for
investigating the aging process. First, reproductive function
is stimulated in quail by daily photoperiods longer than
12 h of light (Foster et al., 1988). Therefore, quail pairs can
remain reproductively active all year in the laboratory.
This characteristic allows direct study of age-related
changes in reproductive function. However, it does raise
the possibility that the lack of a seasonal rest period
accelerates the process of aging or exhausts the reproductive axis. Based on studies in our laboratory with quail,
photoregression of the reproductive system (via short
photoperiod) does not rejuvenate the reproductive system
in birds that are totally senescent (Ottinger and Balthazart,
1986). This method of shutting down the hypothalamicpituitary-gonadal axis at the level of the CNS provides a

useful tool that does not involve surgical gonadectomy or

resulting high gonadotropin levels. Thus, we have utilized
the photocastrated male as a comparison to the aged
senescent males in order to discern age-related functional
changes from those associated with reproductive inhibition
(due to stress or other factors). Second, as described in
more detail below, males show loss of sexual behavior
prior to reproductive failure and females exhibit increasingly irregular egg laying patterns (Ottinger, 1996, 2001;
Holmes et al., 2003). These characteristics provide criteria
useful in separating individuals of the same chronological
age according to reproductive status. Third, females
produce one egg per day, thereby providing an excellent
model of follicular function and rupture and repair
mechanisms (Johnson et al., 1986). Finally, development
occurs without constant maternal influences and sexual
differentiation occurs during embryonic development. As
such, quail are an excellent model for study of the
consequences of early exposure to selected compounds
on long-term reproductive function.
1.1. Aging in male quail
Both males and females show evidence of declining
reproductive function, ultimately leading to reproductive
senescence, with the males living significantly longer
(Ottinger, 1998). Further, all levels of the HPG axis show
age-related decline (Eroschenko et al., 1977; Gorham and
Ottinger, 1986; Ottinger, 1998). Males also show an agerelated decline in fertility coincident with decreasing
reproductive behavior; that precedes measurable loss of
gonadal function. Therefore, reproductive behavior provides an additional, easily monitored index of reproductive
status in males. This has been a useful way of separating
males of the same chronological age into groups of sexually
active or inactive males. Separating males in this manner
revealed that sexually active males retained higher circulating testosterone than sexually inactive males (Ottinger et al.,
1983). In contrast to rats, reproductive behavior is restored
with exogenous testosterone (Ottinger, 1998). There has
also been considerable debate about the issue of aging in
males and if circulating androgen concentrations change.
On balance, there appears to be general agreement that an
age-related decline occurs in males, however gradual
that eventually contributes to reproductive senescence
(Veldhuis, 1997; Ferrini et al., 2001). However, the effects
of declining testosterone have not been well defined in the
male mammal. A recent report documents positive effects of
exogenous testosterone on cognitive performance in aging
male rats (Bimonte-Nelson et al., 2003). Thus, it may turn
out that males experience many of the effects of aging and
declining circulating gonadal steroids on CNS systems,
similar to that observed more precipitously in females.
Similar to mammals, male reproductive behavior is
testosterone dependent and requires aromatization
to estradiol, via the aromatase enzyme (AROM;

M.A. Ottinger et al. / Experimental Gerontology 39 (2004) 16791693

Rosselli et al., 1996a,b; Adkins-Regan and Garcia, 1986;

Watson and Adkins-Regan, 1989a,b; Balthazart et al., 2003).
Further, male copulatory behavior is modulated by the
sexually dimorphic medial preoptic nucleus (POM), which is
an area found to be rich in aromatase enzyme (Panzica et al.,
1991; for reviews Panzica et al., 1996b; Balthazart et al.,
2003). The number of aromatase enzyme (AROM-ir)
neurons and vasotocin immunoreactive (VT-ir) fibers in the
preoptic-septal region, with both neurochemical markers
characterized by a sexually dimorphic distribution (Balthazart, 1997; Panzica et al., 2001) decreased in the POM of
aging males coincident with the loss of sexual behavior;
exogenous testosterone in senescent males restored both
behavior and peptides (Ottinger and Balthazart 1986;
Dellovade et al., 1995; Panzica et al., 1996a). One aim of
the current study was to determine if qualitative or
quantitative differences exist in the cytoarchitecture of
POM and in the AROM-ir cells in the old males that remain
sexually active as compared to males of the same
chronological age that become senescent.
1.2. Aging in female quail
There are notable differences in female reproductive
function in quail including production of an ovum, which
floats in vitellogenin and other components of the yolk
(Ottinger and Bakst, 1995). As the quail hen goes through an
ovulatory cycle every 24 h (produces about 300 eggs/year),
the larger yellow (or yolk filled) follicles are those destined
to ovulate within a few days. Many small white follicles of
various sizes also occur in the reproductive hens ovary. The
follicles contain granulosa and thecal cells that function
similar to mammalian counterparts (Palmer and Bahr,
1992). The smaller follicles produce primarily estradiol,
whereas the larger follicles produce both estradiol and
progesterone. With ovulation, the empty follicle collapses
and undergoes atresia.
During aging, Japanese quail hens have increased loss of
ovarian function and reduced hypothalamic responsiveness,
accompanied loss of cyclicity. This is in contrast to male
quail that undergo gradual reproductive decline. During
aging, hypothalamic response to gonadal steroids
diminishes resulting in a reduced preovulatory LH surge.
Aging females have irregular egg laying, thinning eggshells,
and ovarian regression. Quail maintained on long days
(15L:9D) mature in 810 weeks, maintain peak production
for about 10 months, and then show declining fertility
(3050%) by 70 weeks of age (Ottinger et al., 1983;
Ottinger, 2001; Holmes et al., 2004). Finally, the pattern of
lifetime reproduction and rate of aging is very different in
some slowly aging avian species. These species, such as the
common tern exhibit long life span with continuous
reproductive function (Nisbet et al., 1999, 2002). In these
species, endocrine patterns reflect the maintenance of
reproduction (Ottinger et al., 1995).

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1.3. Age-related decline in hypothalamic responses

In parallel studies we also investigated aging of
hypothalamic systems, with focus on gonadotropin releasing hormone-I (GnRH-I), the molecular species implicated
in regulating reproduction (King and Millar, 1982; Mikami
et al., 1988; Sharp et al., 1990; Dunn et al., 1993; Millam et
al., 1999). Previous immunocytochemical studies in quail
demonstrated that the distribution of GnRH-I producing
neurons is sexually dimorphic (being more numerous in the
male) and strongly sensitive to day-length changes (Foster
et al., 1990). More recently, quantitative double immunofluorescence studies confirmed the strong dimorphism of
GnRH-I neuronal system in the Japanese quail and some of
its peptidergic supply (VT and VIP: Panzica et al., 2001;
CRF: Wang and Millam, 1999). In particular, the number of
GnRH-I cells contacted by VT-containing fibers as well as
the number of GnRH-I cells contacted by VIP-containing
fibers was significantly higher in males than in females
(Panzica et al., 1999).
Functional regulation of the GnRH-I neuron has been
studied in vitro, using parasagittal sections exposed to a
series of secretegogues. The GnRH-I system is responsive to
steroid exposure; furthermore, norepinephrine (NE) stimulated whereas opioid peptides inhibited GnRH-I release
(Li et al., 1994a,b; Fan, 1998). In mammals, many of the
endocrine and behavioral changes associated with aging are
related with falling levels of gonadal steroids (Gruenewald
and Matsumoto, 1991; Tsai et al., 1997). NE and opioid
peptides regulate episodic GnRH release (Dudas and
Mercanthaler, 2001). Conversely, opiate receptor densities
change during aging in response to steroid levels. Therefore,
NE, opioid peptides, and behavioral responses are impacted
by the age-related fall in steroids (Chambers et al., 1981;
Dorsa et al., 1984). Males have a gradual loss in plasma
androgen levels, coincident with declining function of the
GnRH system.
The purpose of these experiments was to further
characterize the age-related changes in hypothalamic
systems that modulate endocrine, neuroendocrine, and
behavioral responses. Therefore, we conducted parallel
studies in which we investigated aging of hypothalamic
systems, with focus on gonadotropin releasing hormone
(GnRH) neurons in both males and females. However,
anatomical and functional changes in the hypothalamus
differed in males and females even with declining
reproductive performance in both sexes. A second objective
was to investigate some of the mechanisms underlying the
age-related loss in reproductive behavior. Our studies had
demonstrated that the aging process in male quail follows a
sequence of loss of reproductive behavior followed by
deteriorating function of the HPG axis. Moreover, the
reproductively senescent male remains responsive to
exogenous testosterone, which restores both sexual behavior
and AROM-ir cell number. This is important as estradiol
production via AROM metabolism is critical to male

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M.A. Ottinger et al. / Experimental Gerontology 39 (2004) 16791693

reproductive behavior as discussed above. Moreover, we

had observed differential individual rates of aging, again
with loss of male reproductive behavior preceding measurable decline in spermatogenesis or steroid production.
However, we did observe changing neuroendocrine system
responses with loss of sexual behavior. Therefore, we
hypothesized that old males that remained reproductively
active would retain AROM-ir cells in the POA-SL region
that regulates reproductive endocrine and behavioral
responses.
In our findings mirror many of the observations in
mammals and strengthen our understanding of the cascade
of changes that accompany declining reproductive function
during aging.

2. Materials and methods

2.1. Experimental animals
A random bred Japanese quail (Coturnix japonica)
colony is maintained at UMCP under IACUC approved
SOPs. Originally, birds were wild caught in Japan and
brought to the USDA, in Dr Howard Opels laboratory,
transferred to Dr Bernard Wentworths laboratory at
University of Wisconsin, and then a breeding population
was established at University of Maryland. Comparison of
our quail with the wild type in Japan indicates that this is
a heterogeneous population, which has retained many of
the wild type characteristics, including smaller body size
and resilience, both in health and reproduction. These
birds are sexually mature in 67 weeks. Birds are
provided feed (Purina Game Bird Startina or Layena)
and water ad lib. They are hatched and brooded for 6
weeks, then transferred to paired animal cages so that egg
production and fertility can be monitored individually. All
aging birds were housed individually after 18 months of
age. The environment is temperature and light (15L:9D)
controlled. Animals and facilities are maintained according to NIH Assurance Statement (A3270-01) under UMCP
IACUC.
The animals for this study were young sexually active
and old sexually inactive of both sexes, and old sexually
active male quail. Reproductive state of females was
assessed by tracking daily egg production; ovarian follicles
(number and stages) and plasma steroid hormones levels
were measured to verify active or non-laying status. In
males, sexual behavior was also used to assess reproductive
status in combination with measurement of the androgen
dependent cloacal gland area and foam production (Ottinger
and Brinkley, 1978). Plasma androgens levels were also
determined along with testes weight at sampling (data not
shown).

2.2. Experimental designs

2.2.1. GnRH-I concentrations and morphology
of the GnRH-I system
Brains were collected fresh frozen (nZ6/sex/age group)
for analysis of GnRH-I and monoamines (measured in the
same microdissected sample). Laying females were
sampled in the morning prior to the preovulatory GnRH-I
surge. Microdissected samples for analyses were taken from
the POA-SL region, which contains the GnRH-I perikarya
and from the posterior hypothalamus and median eminence
(ME) region, which contains the GnRH-I axonal projections
and catecholamine input, especially dopamine (DA) from
the tuberal region. Blood samples were collected from all
birds for later hormone analyses.
2.2.2. GnRH-I elisa-immuno-assay
The competitive EIA uses a mammalian polyclonal
antibody (a gift of Susan Wray, NIH, Bethesda, USA),
which is highly specific for mammalian GnRH and for
chicken GnRH-I, and does not recognize chicken GnRH-II
(Li et al., 1994a,b). Assay sensitivity is 0.01 pg/ml; assay
CV is less than 5%; accuracy is checked with internal
controls. Detailed methods have been published (Li et al.,
1994a,b). Monoamines were measured in the same extract
as GnRH-I assay, using HPLC-EC detection (BAS System,
West Layfeyette, IN); these methods have been published
(Abdelnabi and Ottinger, 2003).
2.2.3. Morphological evaluation using
immunocytochemistry for GnRH-I
Fixed brains were collected from other birds of the same
ages and representing active and inactive reproductive states
(nZ6/age/sex). Birds were anaesthetized and perfused with
4% paraformaldehyde in phosphate buffered saline (PBS,
0.1 M, pH 7.27.4), dissected brains were post fixed and
stored overnight at C4 8C in PBS plus 10% sucrose.
Immunohistochemistry was conducted for GnRH-I, using
an antibody (a gift by Susan Wray, NIH, Bethesda, USA)
titrated to optimize staining and reduce background. This
antibody was validated for cross-reactivity by pretreatment
of control sections pretreated with GnRH-I or GnRH-II
peptides. Sections preincubated with GnRH-I showed no
staining and pretreatment with GnRH-II did not affect
immunostaining. These preliminary data verified the
specificity of this antibody for the GnRH-I form of the
hormone in avian brain. The immunohistochemical procedure is as follows. The entire preoptic-lateral septal region
(POA-SL) was subjected to coronal cryostat sections
(25 mm) into four series, each section within a series was
100 mm apart. One set was thionin stained for anatomy.
Two sets were incubated in primary antibody (1:10,000)
in PBS for 2 days at 5 8C. Sections were then incubated
with a secondary biotinylated antibody, followed by
an avidinbiotinperoxidase complex with Vector SG
substrate (Vector Elite Kit, Burlingame, CA, USA) at

M.A. Ottinger et al. / Experimental Gerontology 39 (2004) 16791693

the manufacturers recommended working dilutions.

Several rinses in PBS were made after each step. Slides
were finally dehydrated, mounted on albumin-gel coated
slides, and coverslipped (Cytoseal XYL, Richard Allen
Scientific, Kalamazoo, MI). GnRH-ir cells were counted in
sections the two series spanning the preoptic-lateral septal
areas (POA-SL) of each animal, using image analysis
software (Image Pro Plus, Media Cybernetics, Silver
Spring, MD, USA).
2.3. Studies on aging of the GnRH-I system: in vitro
perifusion studies
Age-related change in function was assessed in vitro by
perifusion of parasaggittal hypothalamic slices from males
or females of that were young and reproductively active or
old and reproductively senescent.
2.3.1. Perifusion of hypothalamic slices
Parasagittal hypothalamic slices are prepared from a
rectangular block of tissue (POA-SL to ME, lateral to
exclude optic lobes, depth of 1 cm). Anterior-caudal cuts
yield 56 slices of 1 mm thickness. After preincubation in
oxygenated Medium 199 with 5% BSA for 1 h, all slices
from a single brain were placed in a 0.2 ml microchamber
(Endotronics APS 10 Automated Perifusion System; six
chambers) and equilibrated for an additional hour in
Medium 199 with 5% BSA at a flow rate of 12 ml/h.
Following equilibration, NE (10K7 M) was given as a
control in a 10 min pulse followed by a 50 min wash. A
50 min wash is sufficient to remove effects of chemicals in
these experiments. At the end of the experiment there was a
KCl (45 mM) challenge to verify tissue viability. Media
fractions were collected at 5 min intervals and analyzed for
cGnRH-I by EIA. Pulse amplitude and frequency were
analyzed using the Pulsar program (Ramirez et al., 1980).
The validation and preliminary data collected for this
method are published (Li et al., 1994a,b). Experimental
groups were all represented in each run of the six chambers.
2.4. Immunohistochemical study of aromatase
producing system
Previous experimental results had shown that there was a
quantitative loss of aromatase immunoreactive cells
(AROM-ir) in the preoptic-septal region (POA-SL) in old
sexually active or inactive males. Further, old senescent
males treated with testosterone implants showed restored
numbers of AROM-ir cells. Therefore, we conducted two
types of analyses. Size of cells in young, old inactive, and
old inactive testosterone treated males were measured in
Nissl-staining with cresyl violet to determine if there was a
general effect of age and testosterone treatment on cells
in the POM. This analysis was conducted on sections
collected from young, old inactive, and old inactive males
(nZ6/age/treatment group) that had been treated with

1683

testosterone implants for 3 weeks. These implants have

been used in our previous studies and were shown to
restimulate sexual behavior in old inactive males (Ottinger
and Balthazart, 1986; Dellovade et al., 1995; Ottinger et al.,
2002a,b). Second, we conducted a separate experiment in
which young, old inactive, and old active males of similar
age to the inactive males were compared. We hypothesized
that there might be qualitative differences in the AROM
system that allowed old males to remain sexually active. In
this experiment, fixed brains were collected from young,
middle aged, and old sexually active and inactive males
(nZ6/age/group) perfused with Zamboni fixative (4%
paraformaldehyde with 15% picric acid). The frozen brains
were coronally sectioned (30 mm) and alternate sections
were subjected to immunohistochemistry for aromatase
enzyme or Nissl-staining with cresyl violet to verify
anatomical location. A standard peroxidaseantiperoxidase
procedure was employed. Sections were incubated in
primary antibody (rabbit polyclonal antibody raised to
human placental aromatase, 1:1000, see Dellovade et al.,
1995 for more detail) for 48 h at 4 8C. Sections were
incubated with a secondary goat anti-rabbit antibody
(dilution 1/200) followed by incubation in PAP complex
(1/300). The peroxidase activity was finally revealed in a
solution of 0.05 diaminobenzidine with 0.01% hydrogen
peroxide. Sections were then dehydrated and mounted on
slides. In previous studies, we demonstrated that, in the male
quail POM, the cell-size in Nissl-stained sections, as well as
the AROM-ir cell population are differentially sensitive to
testosterone and its androgenic and estrogenic metabolites
in the dorso-lateral and medial subdivisions of the nucleus
(Aste et al., 1993, 1994). Therefore, in the present
experiment we measured the size of Nissl-stained cells, as
well as that of AROM-ir cells, in sections of POM from
young reproductive, old active and old inactive males. Cell
size was measured on Nissl stained sections by image
analysis using NIH-Image 1.55 (a freeware developed by
W. Raysband, NIH, Bethesda, USA) in three sections
containing the medial POM in each individual. Within a
section, quantitative data were collected in six microscope
fields spanning dorso-lateral and medial aspects of the
POM. All neurons, as recognized by a clearly visible
nucleolus and presence of Nissl stain in the cytoplasm in a
field were counted and the program calculated the crosssectional area of each cell. For determining AROM-ir cell
number, one stained section containing the POM at the level
immediately before the anterior commissure was selected
for each bird. Medial and lateral portions of the POM were
identified by reference to the adjacent Nissl-stained section.
A low power image (!5) was captured and AROM-ir cells
were counted within a computer generated grid developed
for the POM. Each cell was identified by the presence of a
cell body and cell body size was calculated using the
thresholding method excluding the neuronal processes.
Statistical analysis of cell size and cell number in these
groups was determined by two-way ANOVA followed,

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M.A. Ottinger et al. / Experimental Gerontology 39 (2004) 16791693

Fig. 1. Concentrations of GnRH-I in the preoptic-septal (POA-SL) and median eminince (ME) regions of young and reproductively senescent male and female
Japanese quail. Asterisk (*) denotes significant difference (p!0.05) within the area with age.

when appropriate, by Fisher PLSD test (Statview 4.1, SAS,

Cary, NC, USA).

age-related decrease was observed in aging senescent

females (Fig. 2).
3.2. GnRH-I system: in vitro perifusion studies

3. Results
3.1. GnRH-I system: in vivo studies
Males and females differed in GnRH-I content in young
adult and aging individuals. In females, the preoptic region
(POA-SL), which contains many of the GnRH-I cell bodies
had comparable average GnRH-I concentrations in young
reproductive hens compared to old non-laying hens (Fig. 1).
In contrast, young females had very high GnRH-I
concentrations in the median eminence (ME), which
contains GnRH-I neuronal projections. Old senescent
males had significantly (p!0.05) decreased GnRH-I
content in both the POA and ME (Fig. 1).
Comparison of the numbers of GnRH-ir cells in young
and old senescent individuals confirmed a sexual dimorphism in young photostimulated adult reproductive individuals
in that males have significantly (p!0.05) more GnRH-I
cells than females. During aging, males experienced a
significant (p!0.05) loss in GnRH-ir cells, whereas no

Perifusion studies provided an experimental paradigm in

which we could compare the response of the GnRH-I system
in individuals of different ages and reproductive states.
Representative graphs showing in vitro GnRH-I release

Fig. 2. Number of GnRH-I immunoreactive cells (GnRH-I-ir) in the POASL of young reproductive and old senescent males and females. Capital
letter denotes significant (p!0.05) difference in young males and females;
lower case letter denotes a significant (p!0.05) decrease in GnRH-I-ir cell
number between young and old males.

M.A. Ottinger et al. / Experimental Gerontology 39 (2004) 16791693

1685

Fig. 3. (a and b) Representative individual graphs of release of GnRH-I (LHRH-I) from parasaggittal hypothalamic slices placed in vitro perifusion. Slices
collected from three young adult reproductive hens and from three old non-laying females. Although these are representative graphs, there were a total six hens
per age group.

from parasaggittal hypothalamic slices collected from three

young and old hens are shown in Fig. 3a and b. Although the
amplitude of GnRH-I peaks tended to decrease, no
significant differences were detected in the amplitude of
baseline or NE stimulated release in females with aging.
However, there were significant differences in basal and NE
challenged GnRH-I release in three young and old males
(Fig. 4a and b). Analysis by Pulsar revealed that, in males,
pulse frequency (Fig. 5a), pulse inter peak interval (Fig. 5b),
and pulse duration (Fig. 5c) did not change during aging.
Rather, the reduced baseline and decreased response to NE
challenge was associated with diminished amplitude of
GnRH-I release resulting in lower cumulative GnRH-I
release (Fig. 5d).
3.3. POM cytoarchitecture and aromatase immunoreactive
system in male quail
Morphometric analysis of Nissl stained sections revealed
a decrease in cell size in both the medial and dorso-lateral

POM in old inactive birds, whereas testosterone treated

males did not show this loss (Fig. 6a). Analysis by one-way
ANOVA for only medial or lateral populations also showed
significant differences (p!0.05 and allowed further analysis
by the Fisher PLSD test. This analysis confirmed a
significant increase in cell size in both lateral and medial
POM cell population of old active males (p!0.05 in
comparison to old inactive; Fig. 6a; Table 1).
Qualitative observation of POM AROM-ir cell population in the two groups of old males (sexually active and
senescent) in comparison to young active animals showed
Table 1
Size of aromatase immunoreactive cells in medial and lateral regions of the
preoptic nucleus

Young active
Old inactive
Old active

Medial POM

Lateral POM

93.72G15.45
99.71G5.57
117.50G13.94

135.58G9.94
124.19G9.36
154.01G23.33

No significant differences were detected.

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M.A. Ottinger et al. / Experimental Gerontology 39 (2004) 16791693

Fig. 4. (a and b) Representative individual graphs of release of GnRH-I (LHRH-I) from parasaggittal hypothalamic slices placed in vitro perifusion. Slices
collected from three young adult reproductive males and from three old senescent males. Although these are representative graphs, there were a total six males
per age group.

that the number of AROM-ir cells decreased during aging in

the old senescent males (Dellovade et al., 1995). This was
also confirmed by further quantitative analysis in the present
experiment (Fig. 6b). Cell number in the dorso-lateral POM
was not significantly affected (F(2,11)Z0.473), whereas it
significantly decreased in the medial subdivision of POM
(F(2,11)Z9.74, pZ0.003), in both old active (p!0.01) and
old inactive (p!0.01) in comparison to young active males,
but with no differences among these two groups. Measurements of AROM-ir cell size also showed a significant effect
on the dorso-lateral population (F(2,11)Z6.5, pZ0.013),
with significantly larger AROM-ir cells in old sexually
active males (p!0.01) than in the other two groups (Fig.
6c). In the medial population, there was a significant effect
(F(2,11)Z5.09, pZ0.027), with smaller AROM-ir cells in
old inactive animals (p!0.01) compared to old active males
(Figs. 7 and 8).

4. Discussion
The interrelationship of endocrine, neuroendocrine and
sexual behavior is intriguing and becomes complex during
the process of reproductive aging. Many studies have
focused on discrete aspects of aging in order to understand
the fundamental biology of the aging process. These studies
have been extremely valuable because they have documented specific neuroendocrine alterations, their impact on
the GnRH system, and on reproductive function, especially
in the female (Wise, 1998, 2000; Wise et al., 1997; Rubin,
2000), and provided the framework for further investigations. We have attempted to be integrative in our
approach by examining aging of neural systems that
modulate and synchronize endocrine and behavioral
components of reproduction. This is one rationale for the
study of the aromatase enzyme system (AROM-ir cells in

M.A. Ottinger et al. / Experimental Gerontology 39 (2004) 16791693

1687

Fig. 5. (ad) Averaged data from in vitro perifusion of parasaggittal hypothalamic slices collected from young and old senescent male Japanese quail. No
difference was found in average GnRH-I pulse inter peak interval (a), pulse frequency (b), or pulse duration (c) in aging males. However, the amplitude of both
basal and NE challenged GnRH-I release were significantly (d; p!0.05) diminished from slices taken from senescent males.

the POA-SL region), which provides sufficient local

metabolisms of testosterone to estradiol to activate and
maintain sexual behavior (Adkins-Regan and Garcia, 1986;
Watson and Adkins-Regan, 1989a,b; Panzica et al., 1991,
1996b; Balthazart, 1997). Age-related changes in neurotransmitter and neuropeptide systems ultimately lead to
reproductive failure. Therefore, although some of the events
occurring during aging may appear disparate, our approach
has been multifaceted in order to identify these fundamental
alterations and their role in the sequence of events in an
aging individual.
4.1. The Japanese quail model for neuroendocrine aging
The Japanese quail exhibits age-related reproductive
decline similar to that documented in mammals. Briefly,
females have increasingly irregular ovulatory cycles with
decreased hypothalamic response to gonadal steroids
(Johnson et al., 1986; Sharp et al., 1992; Ottinger, 1996).
In male quail as in many species, age-related changes in the
quality of sexual behavioral changes herald discernable
endocrine decline (Chambers et al., 1981; Ottinger et al.,
1983; Panzica et al.,1997; Ottinger, 1998). This age-related
decline in reproductive behavior in males does not appear to
be due to a simple loss of gonadal steroids as there was

significant loss of sexual behavior prior to measurable

change in circulating androgens. Rather there appears to be
small changes that ultimately culminate in reproductive
senescence. For example, gonadotropin receptors decrease
in the testes of aging quail a (Chen et al., 2002, 2004;
Ottinger et al., 2002a,b). Supporting data collected in a
number of species and comparison of slowly aging species
to short lived species affords additional insights. For
example, long-lived birds appear to have greater resistance
to oxidative damage (Ogburn et al., 2001). In this paradigm,
the Japanese quail is a model for a rapidly aging avian
species. The Japanese quail also affords the advantages of a
somewhat simpler model, with desirable characteristics for
investigation, including photoperiodicity, neuroplasticity,
and strong sex dimorphism of sexual behavior.
4.2. The GnRH-I system in the Japanese quail during aging
In quail, GnRH-I is one of two forms found in the
hypothalamus (King and Millar, 1982; Sharp et al., 1992).
Our data (see Fig. 2) confirmed the earlier report by Foster
and colleagues (Foster et al., 1990) that there are more
GnRH cells in the male POA-SL region than in the female.
Other studies have implicated GnRH-I as the primary form
of the hormone in the regulation of the reproductive axis

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M.A. Ottinger et al. / Experimental Gerontology 39 (2004) 16791693

Although we did not study the input to the GnRH-I cells

in this set of experiments, previous studies (Panzica et al.,
1997) showed that there were sex differences in the
proximity of GnRH and vasotocin (VT) and vasoactive
intestinal peptide (VIP). However, we have not examined
this relationship during aging. Based on data from the Wise
laboratory, this relationship would be an important facet to
examine in aging females (Wise et al., 1997; Wise et al.,
2002). In addition, other data we have collected provides
evidence for inhibition of GnRH-I release by opioid
peptides even with NE stimulation (Fan, 1998). Moreover,
we found co-localization of d opiate receptors on the GnRHI neuron (Ottinger 2001). These data indicate that
catecholamines and opioid peptides are likely to have
similar roles in the modulation of the GnRH-I system may
be similar among birds and mammals.

Fig. 6. (a) Nissl-stained cell areas in the lateral and medial POM of aging
male quail. There was a significant increase (p!0.05) in cell size during
aging in the medial POM and old inactive males had a significant decrease
(p!0.05) in the lateral POM, which was not observed in the testosterone
treated males. (b) AROM-ir cell number decreased signficantly (p!0.05)
in the medial POM in old males. (c) AROM-ir cell size signficantly
increased (p!0.05) in the lateral and medial POM in aging males.

(Sharp et al., 1990; Millam et al., 1998). Therefore, our

studies focused on the GnRH-I system. While this does not
preclude a physiological role for GnRH-II as has been
hypothesized in other species, such an investigation is
beyond the scope of our studies. Finally, new evidence has
demonstrated the presence of a gonadotropin inhibiting
hormone (GnIH; Ubena et al., 2004). As the actions of this
hormone emerge, it will be very interesting to determine its
role during aging.

4.2.1. Altered GnRH-I content and immunoreactive cell

number in aging quail
There are excellent reviews of the status of the GnRH
system in mammalian female during aging, which
provide literature supporting a clear functional decline
in the response of the GnRH system (Rubin et al., 1984;
Wise et al., 1997; Rubin, 2000; Wise, 2000). However, it
has been difficult for investigators to detect morphological changes in the GnRH system or even altered GnRH
content in the rat model. In contrast, we observed
significantly lower GnRH-I content in the ME region of
aged reproductively senescent (non-laying) hens. The
ovulatory cycle of the quail hen is approximately 24 h
long and the preovulatory luteinizing hormone (LH)
surge occurs 46 h prior to ovulation (Johnson et al.,
1986; Ottinger and Bakst, 1995). In our quail colony,
most young females lay eggs in the late afternoon
(03:0006:00 h). Females in this experiment were
monitored for several weeks to determine daily timing
of egg laying and we sampled prior to the predicted
preovulatory LH surge in order to have individuals at the
same stage of the ovulatory cycle. This would be
expected to relate to greater concentrations of GnRH-I
in the young laying female, presumably at a time of a
substantial releasable pool of GnRH-I. In contrast, the
old reproductively senescent females had sharply reduced
ME GnRH-I content in the old hen. Moreover, both
young and old females had low GnRH-I contents in the
POA, at the site of the GnRH-I perikarya. This sex
difference in GnRH-I content is consistent with the lower
number of GnRH-I-ir cells in females compared to males
(see discussion below).
Data from mammals also demonstrate declining HPG
axis function in the aged male. However, because males
have a gradual decline in reproductive function with
aging, there has been debate about what aspects of
reproductive function decline and when or if reproductive
failure occurs in males (Dorsa et al., 1984; Gruenewald
et al., 1991; Sagrillo et al., 1996; Tsai et al., 1997;

M.A. Ottinger et al. / Experimental Gerontology 39 (2004) 16791693

Lejeune et al., 2003). Our findings in this study showed

sharp decreases in GnRH-I content in the POA-SL and
ME in aging males. It appears likely that the decrease in
the aging male quail may be more definitive than the
parallel process in mammals. One other consideration is
that our studies include extremely old males, which have
become reproductively senescent, including testicular
regression. This may represent an older male, both
chronologically and functionally than is generally investigated in mammalian studies.
Immunohistochemistry confirmed the sexual dimorphisms observed between males and females, where young
reproductive males had more GnRH-ir cells (Panzica
et al., 1995). Further, during reproductive aging, females
retained GnRH-I-ir cells, whereas GnRH-I-ir cell numbers significantly decreased in males. These data are
consistent with observations in female mammals in
which there is little change in GnRH-I-ir cell numbers
even with some more subtle morphological changes
(Rubin et al., 1984; Witkin, 1989; Rubin, 2000; Wise et
al., 2000). In males, the loss of GnRH-I-ir cells is similar
to some of the reports in the male mammal (Dorsa et al.,
1984; Witkin, 1989).
4.2.2. Functional changes in the GnRH-I system
during aging
Some of the most interesting data are in the functional
changes of the GnRH-I system in aging individuals. In
previous studies from our laboratory and other laboratories, functional changes in GnRH release was observed
in vitro with age, reproductive state, and during
photostimulation (Perera and Follett, 1992; Li et al.,
1994a,b). The in vitro perifusion of parasaggittal
hypothalamic slices allowed detection of pulsatile release
of GnRH-I and comparison of NE stimulated release in
hypothalami collected from young reproductive and old
reproductively inactive males and females. These data
confirmed impaired GnRH-I cell response with reduced
baseline and diminished response to norepinephrine (NE).
In females, there was a trend towards lower amplitude of
GnRH-I release in old females, which is similar to some
of the functional changes reported in mammals (Wise et
al., 1997; Gore et al., 2000; Rubin, 2000). Interestingly,
there are data in women that also show decreased GnRH
release and reduced hypothalamic response with menopause, in spite of apparent rising baseline levels of
GnRH (Gill et al., 2002). In our studies, this trend of
decreased baseline and NE stimulated GnRH-I release is
clearly visible in the individual data shown in Figs. 3
and 4, there is greater individual variability in the
females, making it difficult to define an age-related
change. Nonetheless, the avian female appears to have
some alteration in GnRH-I system function, which
translates into increasingly sporadic ovulations and
eventual cessation of the ovulatory cycle and ovarian
failure. The functional basis for the hypothalamic change

1689

during aging may emerge in studies of Fos activation in

GnRH-I cells, which showed reduced number of
activated cells in aging female rats and from coincident
reduced response by the GnRH neuron (Hoffman et al.,
1993; Lloyd et al., 1994; Le et al., 2001). Further, we
have data showing that ovarian function declines later,
after the hypothalamic response has already diminished
(Wu, Holmes and Ottinger, unpublished data). Therefore,
these studies remain of interest and will yield more
insight into the mechanisms of aging in the avian female.
No age-related change in pulse frequency was
observed in either females or males as reported in
pubertal or aging mammals (Hall et al., 2000; Harris and
Levine, 2003). Males did show a significant reduction in
the amplitude of GnRH-I release, both in baseline pulse
amplitude and in NE stimulated GnRH-I release. The
input from NE in stimulating GnRH-I release appears
similar to data from mammals (Dudas and Mercanthaler,
2001). These data clearly demonstrate an age-related
decline in function at the level of the hypothalamus.
Although not directly addressed in this study, it is
important to consider the responsiveness of the hypothalamus to circulating testosterone (Gruenwald and
Matsumoto, 1991). Moreover, it is interesting to relate
this change to reproductive behavior and note that aging
is likely to impact neuroendocrine systems that modulate
both the behavioral and the endocrine components of
reproduction (Chambers et al., 1981; Dorsa et al., 1984;
Tsai et al., 1997; Ottinger, 1998).

4.3. A critical role of gonadal steroids in males

and the aromatase enzyme system
During aging, deteriorating fertility was correlated
with decreased male sexual behavior, and senescent
males lost aromatase immunoreactive (AROM-ir) cells
(Dellovade et al., 1995, and present results). There is
also individual variability in the rate of aging, i.e. males
exhibit delayed reproductive aging while others become
senescent at the same chronological age. Therefore, we
were curious about the potential for neuroplasticity in
these successfully aging males and to ask if there is
evidence for compensatory mechanisms operating during
aging in this species. Further, even old reproductively
senescent males retained some apparent neuroplasticity as
evidenced by restoration of sexual behavior with
exogenous testosterone treatment and the associated
return in AROM-ir cell number. This suggests neuroplasticity of specific neural systems. Finally, aged
sexually active males had fewer, larger AROM-ir cells,
supporting the hypothesis of neuroplasticity of specific
neural systems and verifying the critical role of estradiol
in maintaining reproductive function. We will discuss
these findings in light of mammalian data and relative to
interpretation of these data for functional significance.

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M.A. Ottinger et al. / Experimental Gerontology 39 (2004) 16791693

Fig. 7. Immunohistochemistry for aromatase enzyme (AROM-ir) in the medial preoptic nucleus (POM) in males: young sexually active (a), old sexually active
(b), and old sexually inactive (senescent; c). Old males that remained sexually active had significantly (p!0.05) fewer larger AROM-ir cells.

As in mammals, aromatase enzyme (AROM) has a key

role in modulating male sexual behavior, with reproductive
males having high levels of AROM, presumably to produce
locally high concentrations of estradiol (Balthazart et al.,
1997, 2003). The morphology and anatomical location of
AROM-ir containing cells have been thoroughly described
in mammals and birds and the regulation by estrogen of
aromatase activity may, in part be mediated trans-synaptically (Balthazart et al., 2003). AROM-ir neurons are
surrounded by dense networks of vasotocin-immunoreactive and tyrosine hydroxylase-immunoreactive fibers
and punctate structures. These inputs are partially steroidsensitive and therefore could mediate the effects of steroids
on aromatase activity (for review, see Absil et al., 2001).
Moreover, it is important to understand the relative roles of
the estradiol receptor subtypes in this circuit as these
receptors are known to change in density in various brain
regions with aging and due to stroke damage (Wise, 2000;
Hestiantoro and Swaab, 2004).

The present data suggest that one of these mechanisms

involves plasticity of AROM-ir elements within the POM.
Old males show in fact a decreased number of positive cells
in both POM dorso-lateral and medial subdivisions, but the
cell size of both medial and lateral cell population is larger
in old active male even in comparison to young adult males
(Figs. 7 and 8). This confirms previous results in young
males that showed larger cell size in the lateral cell
population compared to the AROM-ir cells in the medial
POM (Panzica et al., 1991; Aste et al., 1993). These data are
similar to observations on the vasotocinergic innervation of
POM and SL, in which a sharp decrease of VT-ir
innervation occurred in old inactive and photoregressed
males, whereas sexually active adult and old active males
had high levels of VT-ir (Panzica et al., 1996a). Moreover,
there appears to be a clear testosterone action in this
plasticity as evidenced by the data from Nissl-stained
sections. In the medial POM, cell size continued to increase
and testosterone treatment was associated with a difference,

Fig. 8. Immunohistochemistry for aromatase enzyme (AROM-ir) in the medial and lateral preoptic nucleus (POM) in males that are young (6 months of age)
sexually active, old (36 months of age) sexually active, and old (36 months of age) sexually inactive (senescent). Old males that remained sexually active had
significantly (p!0.05) fewer larger AROM-ir cells.

M.A. Ottinger et al. / Experimental Gerontology 39 (2004) 16791693

even compared to old inactive males. Because double

staining was not applied to these sections, we do not know
what cells were larger. Presumably, at least some of these
cells would be the AROM-ir neurons. Further, these
sections revealed restored cell size in the lateral POM
cells in old testosterone treated males to a size found in
young adult males. This change in the old testosterone
treated males did result in a younger morphology, again
involving cells that were not specifically identified via
immunostaining.
Moreover, there appear to be differences in the degree
of plasticity found in young animals compared to that
observed in old males. In young male quail, castration
followed by treatment with testosterone or estradiol
resulted in full recovery in number and cell size of the
AROM-ir system (Aste et al., 1994), whereas in old
active males the AROM-ir cells were less numerous, but
larger in size than in young adult sexually active males.
On the contrary, the VT-ir innervation of POM and SL
were similar for young males and old active males
(Panzica et al., 1995, 1996, 1997). As mentioned earlier,
the capacity for testosterone to restimulate reproductive
behavior and restore an age-related decline in these
neural systems has not been observed in mammals. There
may be several explanations that are pertinent to both the
mammal and bird. First, reduced cell number may be due
to a lack of translation of a particular peptide, in this
case AROM; however, the cells have not died. With
stimulation from exogenous testosterone, the avian brain
may restart the cellular machinery to again produce
AROM. Perhaps, this is one form of neuroplasticity in
that the cells retain the capacity to respond to steroid
hormone stimulation. Second, the apparent compensation
for a loss of some of the AROM-ir cells in old males
that remain sexually active, is also interesting and has
not been reported in this species. The mechanism for the
increased size of the AROM-ir cells may depend on
sufficient signal (testosterone) for the up regulation of
estradiol production in the POA-SL region. Further, it
may be possible to observe these differences in the avian
brain because this species has the capacity to respond to
exogenous steroid hormones with restored neural systems
in areas that modulate and apparently restimulate
reproductive behavior, This apparent neuroplasticity
may reflect phylogenically retained characteristics that
have not been conserved in mammals.
In summary, we have presented data that further
characterizes the age-related decline in neuroendocrine
systems in the Japanese quail model. Many aspects of aging
in this species bear similarities to mammals. However, there
are interesting and important differences in the avian model,
potentially due to neuroplasticity in neuroendocrine systems. As such, this model provides a simpler, advantageous
system in which to investigate selected aspects of the
process of aging.

1691

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