The Japanese quail: a model for studying reproductive aging of hypothalamic systems

Experimental Gerontology 39 (2004) 1679–1693 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

b

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: [email protected] (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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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;

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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 hen’s 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 8–10 weeks, maintain peak production for about 10 months, and then show declining fertility (30–50%) 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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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 Opel’s laboratory, transferred to Dr Bernard Wentworth’s 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 6–7 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 7.2–7.4), 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 avidin–biotin–peroxidase complex with Vector SG substrate (Vector Elite Kit, Burlingame, CA, USA) at

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the manufacturer’s 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 5–6 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

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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 peroxidase–antiperoxidase 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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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.

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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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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

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Fig. 5. (a–d) 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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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 4–6 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 (03:00–06:00 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;

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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

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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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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.

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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.

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