This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yin, W. Articles by Gore, A. C Search for Related Content PubMed PubMed Citation Articles by Yin, W. Articles by Gore, A. C

Neuroendocrine control of reproductive aging: roles of GnRH neurons

¹ Division of Pharmacology and Toxicology, College of Pharmacy and ² Institute for Neuroscience and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA

Correspondence should be addressed to A C Gore at Division of Pharmacology and Toxicology, A1915, University of Texas at Austin; Email: andrea.gore{at}mail.utexas.edu

	Abstract

Top Abstract Overview of reproductive... Evidence for a role... GnRH gene activation and... GnRH release and aging Morphologic changes in GnRH... GnRH neuronal processes and... Inputs to the GnRH... Summary and future directions Acknowledgements References

 
The process of reproductive senescence in many female mammals, including humans, is characterized by a gradual transition from regular reproductive cycles to irregular cycles to eventual acyclicity, and ultimately a loss of fertility. In the present review, the role of the hypothalamic gonadotropin-releasing hormone (GnRH) neurons is considered in this context. GnRH neurons provide the primary driving force upon the other levels of the reproductive axis. With respect to aging, GnRH cells undergo changes in biosynthesis, processing and release of the GnRH decapeptide. GnRH neurons also exhibit morphologic and ultrastructural alterations that appear to underlie these biosynthetic properties. Thus, functional and morphologic changes in the GnRH neurosecretory system may play causal roles in the transition to acyclicity. In addition, GnRH neurons are regulated by numerous inputs from neurotransmitters, neuromodulators and glia. The relationship among GnRH cells and their inputs at the cell body (thereby affecting GnRH biosynthesis) and the neuroterminal (thereby affecting GnRH neurosecretion) is crucial to the function of the GnRH system, with age-related changes in these relationships contributing to the reproductive senescent process. Therefore, the aging hypothalamus is characterized by changes intrinsic to the GnRH cell, as well as its regulatory inputs, which summate to contribute to a loss of reproductive competence in aging females.

	Overview of reproductive senescence in female mammals

Top Abstract Overview of reproductive... Evidence for a role... GnRH gene activation and... GnRH release and aging Morphologic changes in GnRH... GnRH neuronal processes and... Inputs to the GnRH... Summary and future directions Acknowledgements References

 
Aging of the female reproductive system involves all three levels of the reproductive axis, comprising the brain, pituitary and ovary. Most research on the mechanisms of menopause in women has focused on the role of declining ovarian follicular reserves. Although this is unquestionably important, it is becoming increasingly clear from human, nonhuman primate, and rodent models that the hypothalamus and pituitary also play critical roles. Gonadotropin-releasing hormone (GnRH) cells of the hypothalamus are the primary regulatory factor of the reproductive axis (Gore 2002a), driving puberty and ovulation. This primacy of the GnRH system makes it reasonable to postulate that they are also associated with senescent changes in reproductive function, and there is increasing support for such a role. In fact, in the hypothalamus and functionally associated regions, such as the preoptic area (POA), alterations in the GnRH neurosecretory system occur prior to ovarian failure. Nevertheless, the exact mechanisms by which GnRH cells change in terms of their intrinsic properties (e.g. biosynthesis, transport and release), extrinsic regulatory factors (e.g. inputs from central neurotransmitters, neurotrophic factors and steroid feedback) and morphologic changes are only beginning to be understood. In addition, age associated changes in anterior pituitary gonadotrope function, controlling luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion are also implicated in reproductive senescence (Hall & Gill 2001). At the level of the ovary, numbers and viability of follicles; ovulation; and secretion of estrogens, progestins, inhibin and activin change with aging (Burger et al. 2002). Thus, no single level of the hypothalamic-pituitary-ovarian axis can account for the entire process of reproductive senescence. Here, we will address the role of the hypothalamic GnRH neurosecretory system in the loss of reproductive competence in female mammals.

	Evidence for a role of the hypothalamus in reproductive aging

Top Abstract Overview of reproductive... Evidence for a role... GnRH gene activation and... GnRH release and aging Morphologic changes in GnRH... GnRH neuronal processes and... Inputs to the GnRH... Summary and future directions Acknowledgements References

 
Rodents
The depletion of ovarian oocytes is not the primary cause of reproductive senescence in rodents. This was addressed in an early study in which young, reproductively competent rats were ovariectomized, and then given transplanted ovaries from senescent rats. These young recipient animals show follicular development and ovulation from the aged ovaries, demonstrating that the ovary is not the limiting factor in reproductive failure (Krohn 1955). Moreover, although rats become spontaneously acyclic with aging, their ovaries may resume ovulation in response to various external stimuli, and contain functional follicles even in acylic rats (Huang & Meites 1975). Such evidence that rodents become acyclic in the presence of a functional ovary indicates a nonovarian, putative hypothalamic or pituitary signal that plays a causal role in reproductive failure.

Further support for a role of the hypothalamus is provided by studies utilizing electrolytic lesions of the medial POA in young rats. These animals show irregular estrous cycles as in older rats (Clemens & Bennet 1977). Electrochemical stimulation of the POA of aged acyclic rats results in enhanced LH secretion (Wuttke & Meites 1973). This latter finding indicates that the pituitary continues to be responsive to GnRH release (which is elicited by the POA stimulation) and suggests that altered GnRH output, not pituitary responsiveness, is responsible for age-related acyclicity.

Primates
There are clear species differences in reproductive aging processes. As discussed above, the hypothalamus probably plays the primary role in reproductive senescence in rodents. However, the relative roles of the hypothalamus, pituitary and ovary in both human and nonhuman primates are less clear, although all three of these levels appear to contribute, at least to some extent, to the reproductive aging process. A study in female rhesus monkeys shows that pulsatile GnRH release (specifically mean GnRH concentrations and to a lesser extent GnRH pulse amplitude) becomes elevated very late in life (28–31 years) (Gore et al. 2004), concomitant with the perimenopause in this species. Studies in women have measured serum gonadotropin (LH or FSH) or glycoprotein free -subunit (FAS), the latter as a reflection of hypothalamic GnRH release (Gill et al. 2002) to determine age-related changes. One study compared pulsatile gonadotropin release (frequency and amplitude) in postmenopausal women relative to premenopausal levels during the early follicular stage and showed that the postmenopausal women had increased pulse amplitude, but no change in pulse frequency, compared with the premenopausal cohort (Rossmanith 1995). However, because the premenopausal women were sampled during the early follicular stage, when pulsatile gonadotropin release is relatively low, it is difficult to generalize about increased gonadotropin release between pre- and postmenopausal women. More insight is provided by studies comparing early and late postmenopausal women. The study by Rossmanith (1995) shows that, as aging progresses, gonadotropin levels decrease, as do gonadotropin pulse frequency and amplitude. Another group compared early postmenopausal women (45–55 years) with late postmenopausal women (70–80 years) and demonstrated significantly lower LH and FSH concentrations in older than younger postmenopausal women (Gill et al. 2002). A second report from this group shows that FAS pulse frequency is significantly decreased, LH pulse frequency is modestly decreased and both FAS and LH pulse amplitude are lower in the aged group (Hall et al. 2000).

It could be argued that because the perimenopause is characterized by decreased circulating estradiol concentrations relative to the premenopausal period (Gore et al. 2004), the increased GnRH/gonadotropin secretion seen in monkeys (and possibly in early postmenopausal women) (Rossmanith 1995) is simply due to a diminution of estradiol negative feedback. However, this is probably not entirely the case, suggesting a hypothalamic and/or pituitary level of regulation. Again, studies comparing early and late postmenopausal women have shed light on this phenomenon. Estradiol levels are not significantly different between early and late postmenopausal women (Hall et al. 2000), yet serum LH, FSH and FAS levels are significantly lower in these late postmenopausal women (Hall et al. 2000, Gill et al. 2002), and gonadotropin negative feedback regulation by exogenous steroids is maintained (or even increased) in late postmenopausal women (Gill et al. 2002). A recent paper on the SWAN study (Study of Women’s Health Across the Nation) (Weiss et al. 2004) shows that perimenopausal women at about 50 years of age who exhibit cyclic changes in urinary estrone conjugates (a reflection of estrogens) across a menstrual cycle can be subdivided into two groups: those with LH profiles consistent with a preovulatory LH surge, and those with no LH response. Although this finding is not entirely consistent with earlier work, it supports a hypothalamic and/or pituitary loss of sensitivity to estrogen feedback in women, and not a change in ovarian output per se, as being a primary initiator of the menopausal process.

To follow is a summary of the literature on the role of the hypothalamic GnRH neurosecretory system in reproductive aging. Our focus will be GnRH functional, morphologic and anatomic properties that correlate with, or may cause, reproductive failure. We will also discuss briefly some of the regulatory factors controlling GnRH neurons that themselves undergo age-related changes, the end result of which is the process of reproductive senescence.

	GnRH gene activation and aging

Top Abstract Overview of reproductive... Evidence for a role... GnRH gene activation and... GnRH release and aging Morphologic changes in GnRH... GnRH neuronal processes and... Inputs to the GnRH... Summary and future directions Acknowledgements References

 
The synthesis of the GnRH decapeptide, like other secreted peptides, begins with the transcription of the GnRH gene to an RNA primary transcript in the nucleus. After processing, including 5' capping, polyadenylation and intron splicing, the mature GnRH mRNA is translocated to the cytoplasm. Relative to most genes, the GnRH mRNA precursors in the nucleus are relatively abundant in vivo (Gore 2002b). This led us to hypothesize that there is a large pool of GnRH mRNA precursors, enabling most of the regulation of GnRH biosynthesis to occur primarily by a post-transcriptional mechanism such as mRNA stability (Gore 2002b). Once the mRNA is produced, translation of GnRH occurs in the endoplasmic reticulum and Golgi apparatus, and ultimately via budding of secretory vesicles, which contain the GnRH precursor peptide. Processing of the GnRH precursor peptide continues in the vesicles, which contain the enzymes involved in proGnRH peptide splicing. Thus, within vesicles, both the 10-amino-acid GnRH and its 56-amino-acid GnRH-associated peptide (GAP) can be detected, and both are released from nerve terminals in the median eminence (Rangaraju et al. 1991, Wetsel & Srinivasan 2002). The former exerts its hypophysiotropic actions at the pituitary, and the latter has functions that are only partially understood.

GnRH gene expression in ovarian-intact models
GnRH mRNA levels are measured as an index of biosynthetic capacity. In ovarian-intact Sprague-Dawley female rats, used either on proestrus or diestrus (the two extremes of the estrous cycle), our group used the RNase protection assay to show that overall GnRH mRNA levels are significantly higher in middle-aged (12–14 months) and old (25–26 months) than young (4–5 months) rats (Gore et al. 2000a) (Table 1). In these same animals, GnRH primary transcript levels, an index of gene transcription, are significantly lower in old than young or middle-aged rats (Gore et al. 2000a). This finding suggests that the age-associated increase in GnRH mRNA occurs independently of de novo gene transcription; in fact, the increase in mRNA occurs despite a decrease in gene transcription. Another study in Sprague-Dawley rats shows a small (20%) age-related decrease from 2 to 18 months of age, but that study did not take reproductive cyclicity into consideration (Li et al. 1997b). Age differences may explain the differing results between these two rat studies. It is also important to point out that ovarian-intact rats, in contrast to humans, may still possess functional ovaries that are capable of responding to external stimuli with ovulation even at very advanced ages (Huang & Meites 1975). Rance’s laboratory used in situ hybridization to show that GnRH mRNA levels are significantly higher in postmenopausal than in premenopausal women, the latter of varying menstrual cycle status (Table 1) (Rance & Uswandi 1996). The similarity of Rance’s finding in women to our finding in rats (Gore et al. 2000a), that is, age-associated increases in GnRH mRNA levels, suggests that, whereas the ovarian environment is very different, the hypothalamic level may be conserved across mammalian species.

GnRH gene activation during the preovulatory GnRH/LH surge of intact rats
One of the hallmarks of reproductive aging in rats is the attenuation of the preovulatory GnRH/LH surge that is responsible for ovulation (Lloyd et al. 1994). Therefore, this is an important endpoint at which to study GnRH gene expression. To address this, we euthanized young and middle-aged proestrous rats at 1000 and 1500 h. These time points were chosen as we previously showed in young female rats that GnRH mRNA and primary transcript levels are elevated at 1500 compared with 1000 h on proestrus, the day of the surge (Gore & Roberts 1995). This latter finding was recently confirmed in young rats by another laboratory using real-time PCR (Schirman-Hildesheim et al. 2005). In our study comparing young and middle-aged intact rats during the proestrous gonadotropin surge, we showed that GnRH mRNA levels are higher at 1500 than 1000 h. A trend for the GnRH primary transcript to be increased at 1500 compared with 1000 h was also seen in young rats. By contrast, middle-aged rats lack an increase in GnRH mRNA or primary transcript on proestrus (Gore et al. 2000a) (Table 1). This lack of drive of the GnRH system of middle-aged rats may explain the delay and attenuation of the preovulatory GnRH/LH surge.

The immediate early gene Fos has been used for many years as a marker of transcriptional activation (reviewed in Hoffman & Lyo 2002). In GnRH cells, Fos coexpression is upregulated within GnRH neurons of young rats during the preovulatory GnRH/LH surge (Lloyd et al. 1994). This implies an activation of GnRH gene expression at this time, and is consistent with our report, discussed above, that GnRH gene transcription is elevated shortly before the surge in young female rats (Gore & Roberts 1995). In proestrous middle-aged rats whose preovulatory surge is attenuated, Fos coexpression in GnRH neurons is substantially decreased compared with young proestrous rats (Lloyd et al. 1994, Le et al. 2001) (Table 1). Again consistent with this, we reported that GnRH primary transcript RNA levels in middle-aged rats do not increase on the afternoon of the GnRH/LH surge, implying a loss of drive of GnRH gene activation in middle-aged rats, even those which are still exhibiting a surge and can ovulate. Thus, this change precedes the transition to acyclicity and may play a causal role in this process.

GnRH gene activation during the steroid-induced GnRH/LH surge of OVX rats
In OVX rats, GnRH gene expression has been quantified by in situ hybridization during the steroid-induced surge in young and middle-aged rats (Rubin et al. 1997). The authors show an increased number of GnRH mRNA-expressing neurons at 1800 h (near the peak of the surge) in young, but not middle-aged, animals. Le et al.(2001) studied OVX young and middle-aged rats during the estradiol plus progesterone-induced surge, and show that Fos coexpression in GnRH neurons is significantly lower in middle-aged than young rats.

Taken together, these studies support the importance of measuring age-related changes in GnRH gene expression, as they relate to the functional outcome of reproductive failure. Studies performed during the preovulatory or steroid-induced GnRH/LH surge have been particularly informative in this regard, as they consistently support a diminution of the activation of surge-associated GnRH gene activity by middle age, prior to reproductive failure (Table 1).

	GnRH release and aging

Top Abstract Overview of reproductive... Evidence for a role... GnRH gene activation and... GnRH release and aging Morphologic changes in GnRH... GnRH neuronal processes and... Inputs to the GnRH... Summary and future directions Acknowledgements References

 
After synthesis, GnRH is packaged into secretory granules and transported to the median eminence, where the peptide is released from GnRH terminals into the perivascular space of the portal capillary system (King et al. 1982). In adult rodents, GnRH is released from the median eminence at a frequency of about one pulse every 30 min. A slightly slower frequency of release (approximately 50–60 min intervals) is observed in primates (Terasawa 2001, Gore et al. 2004). The pulsatile pattern of GnRH (or LH) release is superimposed upon longer cycles of release, such as menstrual (primates) or estrous (rat, mouse, sheep) cycles. During aging, all of the parameters of pulsatile release (basal and mean concentrations, and pulse frequency) and the preovulatory surge are altered (as described below and summarized in Table 2). As all of these biologic rhythms are critical for normal reproductive function (Levine 1997, Terasawa 2001), age-related changes in these patterns may underlie the transition to acyclicity. To follow is a summary of in vivo and in vitro studies on GnRH or LH release in aging females.

In vivo studies of the preovulatory GnRH/LH surge in intact rats
LH release has also been measured during the preovulatory GnRH/LH surge, which is temporally delayed and attenuated in middle-aged rats (Lloyd et al. 1994, Rubin et al. 1994, Le et al. 2001) (Table 2), despite the fact that these animals can still have regular reproductive cycles, ovulate, and become pregnant. Nevertheless, this diminution of preovulatory GnRH/LH release, which is correlated temporally with diminution in the proestrous increase in GnRH gene expression (Gore et al. 2000a), seems to presage the transition to acyclicity. Indeed, both positive and negative feedback regulation of GnRH/LH release become compromised with aging in rats (Huang et al. 1976). Although the mechanisms for the surge attenuation and the diminished response to estradiol feedback are not well understood, they probably involve changes in the capacity of GnRH neurons to synthesize and release the decapeptide, as well as to respond to neural and glial inputs and steroid hormone sensitivity.

In vivo studies of the steroid-induced GnRH/LH surge in OVX rats
Direct measurements of GnRH release by push-pull perfusion in aging female rats show a decrease in pulsatile GnRH release during a steroid-induced LH surge in middle-aged compared with young OVX rats (Rubin & Bridges 1989). Specifically, mean GnRH levels and pulse frequency (but not pulse amplitude) are diminished, and mean GnRH output during the surge is lower in middle-aged rats (Table 2). Sano and Kimura (2000) measured multiunit activity (MUA) in the median eminence of young (3 months) and old (26 months) OVX rats, together with serial serum LH measurements, as a reflection of GnRH activity. They showed that both MUA and LH frequency decrease, and LH pulse amplitude is smaller, in the aged rats. These findings of decreased GnRH release and hypothalamic MUA are consistent with the age-related changes in LH release in rats discussed above.

In vivo studies in primates
GnRH and LH release have been measured in aging rhesus monkeys undergoing menopause by Woller et al.(2002) and Gore et al.(2004) (Table 2). Both of these studies demonstrate the maintenance of robust GnRH and LH pulses in aged animals, with overall elevated pulse amplitudes but no change in pulse frequency compared with young monkeys sampled during the early follicular phase. Our study measuring GnRH release in monkeys also reports large GnRH bursts in some of the old animals (Gore et al. 2004), and we speculated that these incidents may be related to hot flashes. Japanese macaques show elevated serum LH as they age, and continue to exhibit positive feedback responses to estradiol treatment (Nozaki et al. 1995). These results contrast markedly with those in rats discussed above that show a decrease in pulsatile GnRH and LH release and a loss of positive feedback to estradiol with age. However, rats and monkeys differ in the age at which they undergo reproductive senescence. Rats experience this transition in midlife, whereas nonhuman primates, such as rhesus monkeys, undergo this process much later in life. Some light may be shed on this issue by studies measuring serum gonadotropins or glycoprotein free -subunit (FAS) in women as an index of GnRH release (Table 2). Although these studies differ in the age of experimental subjects, overall they show increases in gonadotropin concentrations from the pre- to the perimenopausal or early postmenopausal period (Rossmanith 1995, Gill et al. 2002), but subsequent decreases in gonadotropin or FAS release from the early to the late postmenopausal period, including lower hormone concentrations and decreased pulse frequency (Ushiroyama et al. 1999, Hall et al. 2000). Thus, the chronologic age relative to reproductive failure is an important parameter in understanding species differences.

In vitro release of GnRH
Studies of GnRH release from explanted hypothalamic or median eminence dissections have provided some information on how these cells change their function during aging. These experiments have the advantage of facilitating direct measurements of GnRH, although they also have some caveats, as described below. In intact (proestrous), OVX, or OVX plus estradiol-treated female rats (the latter during the LH surge), Rubin (1992) reported that basal GnRH release is higher from isolated hypothalami of middle-aged than young rats, although potassium-stimulated GnRH release is equivalent between the two ages. A different result was reported by Hwang et al.(1990), who demonstrated that basal GnRH release from explants of young (~4 months) and old (21–24 months) OVX rats is comparable, whereas potassium-stimulated GnRH release is greater in the young rats. Differences between these two reports may be due to differences in ages of animals, as the Hwang study used much older rats than the Rubin study. Another group showed no difference in basal GnRH release, but a decrease in stimulated GnRH release caused by glutamate agonists in middle-aged proestrous compared with young proestrous rats (Zuo et al. 1996). Although some of these inconsistent results may be attributable to differences in the ages at which rats were studied, others may also result from properties of explants, which lack a functionally intact GnRH network. Thus, if explants are cut or prepared differently between laboratories, this may result in differential GnRH neural inputs that might influence basal and/or stimulated GnRH release. Therefore, while results in explants have provided some valuable information, the more difficult in vivo studies are more relevant to age-related changes in GnRH release.

	Morphologic changes in GnRH neurons with age

Top Abstract Overview of reproductive... Evidence for a role... GnRH gene activation and... GnRH release and aging Morphologic changes in GnRH... GnRH neuronal processes and... Inputs to the GnRH... Summary and future directions Acknowledgements References

 
According to the evidence above, the nature of GnRH neuronal activity changes with age. This change may be related to alterations in numbers of GnRH cells and/or their morphology, reflecting the manufacture, transport and release of GnRH.

The question of whether aging affects GnRH cell numbers has been pursued at the light-microscopic level. As summarized in Table 3, the results vary, possibly in relation to the different treatments, ovarian status and species. Nevertheless, most studies report either no age-related change or a small difference in cell numbers with aging. An increase in GnRH cell numbers in old (constant estrous) compared with young (proestrous) rats was reported by one group (Merchenthaler et al. 1980). Several other investigations report no change in GnRH cell numbers, distribution or morphology with aging in female rats (Rubin et al. 1984, Miller & Gore 2002), mice (Hoffman & Finch 1986) or rhesus macaques (Witkin 1986). Still other studies have reported an age-associated decrease in GnRH cell numbers in mice and rats (Miller et al. 1990, Funabashi & Kimura 1995), but the detected decreases were modest (<20%). Therefore, the majority of studies suggest that the total number of GnRH neurons may not be a critical parameter in the determination of reproductive senescence.

In the POA, the relationship between GnRH perikarya and astrocytes may change with aging. Cashion et al.(2003) evaluated the number of astrocytes that are apposed to GnRH neurons in young and middle-aged cycling rats across different times on proestrus and estrus. They showed that glial apposition to GnRH neurons undergoes circadian fluctuations on proestrus (but not estrus) in young, but not middle-aged, rats, and suggest that this loss of astrocytic plasticity may contribute to the delayed and attenuated LH surge in middle-age rats.

GnRH neuronal ultrastructure undergoes age-related alterations, as shown by electron-microscopic studies and described in Table 3. By pre-embedding immunohistochemistry, GnRH-positive cell bodies are identified as containing a large round or ovoid nucleus, well-developed rough endoplasmic reticulum (RER), Golgi complexes, lysosomes, scattered dense core vesicles characteristic of peptidergic vesicles (100 nm in diameter), and small clear vesicles characteristic of monoaminergic neurotransmitters (30–40 nm) (Jennes et al. 1985). A large proportion of the surface area of GnRH perikarya appears to be covered by a thin glial sheath, which separates the plasma membrane from other axon processes (Jennes et al. 1985, Witkin et al. 1991). Reports on the effects of age on the ultrastucture of GnRH neurons have focused on synaptic inputs, the volume fraction of RER and the Golgi apparatus (Table 3). In intact rats, RER and Golgi volume fractions are significantly lower in GnRH cells of old, acyclic rats (18–20 months) than in those of young, cycling rats (4 months, used on diestrus). In similarly aged rats that have been OVX for 4 weeks, the percentage of Golgi fraction is lower in aged than young rats, but the percentage of RER is much higher in the old rats. These results suggest that aging is associated with changes in protein synthesis of GnRH cells with aging in an ovarian status-dependent manner (Romero et al. 1994). In addition, these studies on GnRH ultrastructural changes, showing decreases in protein biosynthetic machinery, are consistent with some of the changes observed in GnRH gene expression and release discussed above.

	GnRH neuronal processes and terminals change with aging

Top Abstract Overview of reproductive... Evidence for a role... GnRH gene activation and... GnRH release and aging Morphologic changes in GnRH... GnRH neuronal processes and... Inputs to the GnRH... Summary and future directions Acknowledgements References

 
Relatively few studies have investigated the question of age-associated changes at the level of GnRH neuronal processes and terminals. Nevertheless, it is important to perform studies at this level of analysis, and to contrast results with effects of age on the perikarya. At the cell body, the age-related changes discussed above in synaptic input or intracellular organelles would probably affect GnRH biosynthesis. By contrast, at the GnRH nerve terminal, as discussed below, age-related alterations would directly affect GnRH neurosecretion. Moreover, neuronal or nonneuronal elements regulating GnRH nerve terminals in the median eminence may change with aging to result in altered GnRH neurosecretion.

GnRH processes project caudally through the hypothalamus, with their terminals ultimately projecting to the external zone of the median eminence. At the light-microscopic level, GnRH axon swellings (Herring bodies), often referred to as puncta or varicosities, can be easily identified by immunocytochemistry. At the electron-microscopic level, these structures are seen to contain clusters of large dense-core and small vesicles in GnRH processes and, rarely, to form synaptic contacts with other neurons. Besides having a storage function, the axon swellings may be a site of neuronal interaction (Jennes et al. 1985, Tweedle et al. 1989). A few studies have investigated changes in GnRH terminal morphology in the context of aging (Table 3). There is reduced GnRH fiber staining in the median eminence of old male rats (Hoffman & Sladek 1980). In intact female rats, GnRH immunoreactivity is decreased in the median eminence of young compared with middle-aged female rats during the preovulatory GnRH/LH surge (Rubin & King 1995), suggesting that greater GnRH release occurred from the terminals of the young rats (Table 3). Using light microscopy, Bestetti et al. found that the number, area and immunoreactivity of GnRH axons in the median eminence are decreased in old compared with young rats (Bestetti et al. 1991).

Electron-microscopic analyses of terminal regions of young rats show that the distance between GnRH terminals and the basal lamina, the ensheathment of GnRH terminals by tanycytes, and the proximity of GnRH terminals to portal capillaries are affected by sex, developmental age and hormonal status (King & Rubin 1994). Considerable regulation of GnRH terminals may result from ensheathment by tanycytes (specialized ependymal cells lining the third ventricle) (Flament-Durand & Brion 1985), which could play a role in allowing GnRH terminals access to the portal vasculature or to other neuronal processes. Tanycytes are believed to be involved in the transport and release of hormones, including estrogen and GnRH, in the median eminence (Akmayev & Fidelina 1981). It has also been proposed that tanycytes may play a role in the remodeling of the aged basal hypothalamus in male and female rats through phagocytotic actions on degenerating neurons (Brawer & Walsh 1982, Zoli et al. 1995). Light- and electron-microscopic studies show that tanycytes undergo morphologic modifications with age in both male and female rats (Brawer & Walsh 1982). Variations of estrogen and neurotransmitter levels in the median eminence during reproductive cycles have been shown to affect the morphologic, synthetic and transport activity of tanycytes (Flament-Durand & Brion 1985, Zoli et al. 1995). Therefore, the relationship between GnRH terminals and tanycytes is a level of regulation that appears to be affected during aging, and has potential consequences for GnRH release into the portal capillary circulation.

GnRH is present in secretory granules within axon profiles and nerve terminals of the median eminence (Goldsmith & Ganong 1975, Yin et al. 2002). The size of the granule is smaller in the portal plexus (40–70 nm) than in the GnRH process itself (90–120 nm) in adult male guinea pigs (Silverman & Desnoyers 1976). Recently, immunogold labeling has been applied to study the GnRH network in rat (Liposits et al. 1995, Prevot et al. 1998) and rhesus monkey (Durrant & Plant 1999). With cryofixation, low temperature embedding and quantitative immunogold labeling techniques at the electron-microscopic level, it is possible to probe the subcellular localization of a neurotransmitter or protein within a very small compartment, linking function with morphology. We have been applying this technique to understand the ultrastructural regulation of GnRH terminals by estrogen (Yin et al. 2002) and are currently extending our analyses to aging rats.

	Inputs to the GnRH system change with age

Top Abstract Overview of reproductive... Evidence for a role... GnRH gene activation and... GnRH release and aging Morphologic changes in GnRH... GnRH neuronal processes and... Inputs to the GnRH... Summary and future directions Acknowledgements References

 
The regulation of pulsatile GnRH/LH release and the preovulatory surge involves multiple neuronal circuits (Gore 2002a, 2002b). Although the number of neurotransmitters and neuromodulators is too large to cover in the present review, we have summarized information on a few of the major neurotransmitters commonly believed to regulate GnRH function and for which there is evidence of age-related alterations, the outcome of which is a diminution of reproductive competency.

Dopamine
In the aging hypothalamus of male and female rats, dopamine (DA) secretion into the portal vasculature declines (Gudelsky & Porter 1981, Reymond & Porter 1981). The release of DA from tuberoinfundibular dopaminergic (TIDA) neurons into hypophysial portal blood vessels is sexually dimorphic, and related to the stimulatory action of estrogen (Gudelsky & Porter 1981). The importance of DA in reproductive function has been shown by the ability of dopaminergic agonists to restore estrous cycles in acyclic rats, either young (with lesions in medial POA) or aged (Clemens et al. 1977). In male rats, DA fluorescence intensity decreases in the median eminence of old compared with young rats, but increases with age in the dopaminergic perikarya of the arcuate nucleus (Hoffman & Sladek 1980). Morphologic evidence shows a rapid and dramatic decrease in the total number of neurovascular contacts in the median eminence after DA infusion into the lateral ventricle (Hokfelt 1973). DA and cAMP regulated phosphoprotein (DARPP-32, a marker for cells containing DA receptors) show strong immunoreactivity in tanycytes surrounding GnRH neuroterminals in the median eminence of male rats and monkeys (Meister et al. 1988). These results suggest that DA may control GnRH release in the median eminence by modifying the degree of tanycytic enclosure on GnRH terminals (Meister et al. 1988).

Norepinephrine
Norepinephrine, acting primarily through the -adrenergic receptor, stimulates GnRH release, and this noradrenergic influence declines with aging (Temel et al. 2002). Using young and middle-aged OVX rats during the steroid-induced gonadotropin surge, Wise (1982a) showed that norepinephrine concentrations in several hypothalamic regions are elevated on the afternoon of the surge in young rats, but that this is attenuated in middle-aged rats. There is a potential anatomic basis for this decreased responsiveness with aging, as a study reported direct appositions between GnRH and noradrenergic cells in young (5 months), but not old (24 months), female OVX mice (Miller & Zhu 1995). Clonidine, an -adrenergic agonist, can restore the pulsatile pattern of LH secretion in middle-aged acyclic rats (Estes & Simpkins 1982). Therefore, changes with aging in the anatomic and functional relationships between noradrenergic and GnRH neurons may contribute to the decline in reproductive function.

Glutamate
Abundant evidence indicates that the excitatory neurotransmitter glutamate is extremely important in the regulation of GnRH function. GnRH cells express glutamate receptors (both N-methyl-D-aspartate (NMDA) and non-NMDA receptors) at their cell bodies and nerve terminals (Gore et al. 1996, Kawakami et al. 1998, Miller & Gore 2002). In POA of intact (young, middle-aged and old) rats, mRNA levels of NMDA receptor subunits change with aging (Gore et al. 2000b). Overall levels of the NMDA receptor 2b (NR2b) subunit decrease, not with chronologic age but rather with reproductive status. Thus, young and middle-aged rats that are regularly cycling (used either on proestrus or diestrus I) have higher NR2b mRNA levels in the POA than do middle-aged acylic or old acylic rats. In addition, the coexpression of NR2b specifically on GnRH perikarya increases in intact middle-aged (either proestrous or persistent estrous) compared with young, proestrous rats, suggesting an alteration in the NMDA receptor stoichiometry with aging, but largely independent of reproductive cycling status (Miller & Gore 2002). This probably has a functional outcome, as the ability of NMDA agonists to stimulate GnRH gene expression in young proestrous rats is abolished in middle-aged proestrous or persistent estrous rats (Gore et al. 2000b). Additionally, the stimulatory effect of NMDA on GnRH release is diminished in old (18 months) compared with young (3 months) OVX rats (Arias et al. 1996). Finally, new evidence shows that GnRH neurons of male rats coexpress vesicular glutamate transporter-2 (VGLUT2), the presence of which indicates a glutamatergic phenotype (Hrabovszky et al. 2004). This suggests that GnRH neurons themselves may synthesize and potentially release glutamate. To date, this has been reported only for male rats; it will be interesting to extend this finding to females, and to determine whether this coexpression changes with age.

Vasoactive intestinal polypeptide
The suprachiasmatic nucleus (SCN), the circadian pacemaker in the brain, makes projections to GnRH neurons that are critical for circadian (daily) cycles of GnRH release (Krajnak et al. 1998). A strong candidate for the regulation of GnRH neurons by the SCN is vasoactive intestinal polypeptide (VIP). VIP neurons project directly to GnRH neurons, where they make direct contacts (Van der Beek et al. 1997) in a sexually dimorphic manner, and there are more contacts in female than male rats (Horvath et al. 1998). VIP neurons undergo developmental and age-related changes in their circadian rhythms, and in their inputs to GnRH perikarya (Krajnak et al. 1998, Kriegsfeld et al. 2002). VIP mRNA levels exhibit a 24 h rhythm in young, but not middle-aged, female rats, which is associated with declines in cellular VIP mRNA and VIP neuron number in SCN (Krajnak et al. 1998). It has been suggested that the effects of VIP on GnRH neuron are mediated through cAMP. A study of cAMP level in SCN and POA (the location of GnRH perikarya) demonstrates a diurnal rhythm in young, but not middle-aged, female rats (Gerhold et al. 2005). When the VIP rhythm is suppressed, this produces an aging-like cAMP rhythm, and in turn leads to inhibition of the GnRH activation (Gerhold et al. 2005).

Insulin-like growth factor-I
Insulin-like growth factor-I (IGF-I) is a neurotropic factor in the brain, where it is involved in neuronal development. IGF-I receptors (tyrosine kinase receptors) are widely distributed in the brain, including the hypothalamus and POA (Daftary & Gore 2005). Recently, we showed that IGF-I immunoreactivity is coexpressed in GnRH cell bodies of intact male and female mice (female mice were randomly cycling), and that IGF-I immunoreactivity is abundantly expressed in regions enriched with GnRH nerve processes and terminals (Daftary & Gore 2003, 2005). IGF-I mRNA levels are substantially lower in both the POA (the location of GnRH perikarya) and medial basal hypothalamus-median eminence (the site of GnRH processes and terminals) of middle-aged than young OVX rats (either with or without estradiol replacement) (Miller & Gore 2001). The finding that IGF-I treatment does not affect GnRH gene expression in rats of either age suggests that it may act on other levels of the GnRH circuit rather than gene expression (Miller & Gore 2001).

	Summary and future directions

Top Abstract Overview of reproductive... Evidence for a role... GnRH gene activation and... GnRH release and aging Morphologic changes in GnRH... GnRH neuronal processes and... Inputs to the GnRH... Summary and future directions Acknowledgements References

 
Reproductive function declines with aging in female mammals, and the contribution of the hypothalamic GnRH system to this process is becoming better understood. Studies on how the GnRH neurosecretory system changes with aging have demonstrated alterations in GnRH gene expression, the pattern of pulsatile and preovulatory GnRH release, and the morphology and ultrastructure of the GnRH cell body and neuroterminal. Neurotransmitters and neuromodulators also show important regulatory effects on the GnRH circuit, and these, too, can undergo age-related alterations. The use of advanced molecular and structural biology techniques may provide us with improved resolution and a broader perspective on the study of GnRH neural activity in the future. Although this review has limited itself to changes within GnRH cells and their endogenous regulatory factors in the brain, it is important to consider the influence of the environment on the biosynthesis of GnRH. Interactions among GnRH neurons, environmental factors and genes will be fruitful areas for further studies on the mechanisms of reproductive aging.

	Acknowledgements

Top Abstract Overview of reproductive... Evidence for a role... GnRH gene activation and... GnRH release and aging Morphologic changes in GnRH... GnRH neuronal processes and... Inputs to the GnRH... Summary and future directions Acknowledgements References

 
We are grateful to the National Institutes of Health (grant no. AG16765 to A C G) for support of the work described herein. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

	Footnotes

 
Received 13 July 2005
First decision 2 September 2005
Revised manuscript received 14 November 2005
Accepted 22 November 2005

	References

Top Abstract Overview of reproductive... Evidence for a role... GnRH gene activation and... GnRH release and aging Morphologic changes in GnRH... GnRH neuronal processes and... Inputs to the GnRH... Summary and future directions Acknowledgements References

 

Akmayev IG & Fidelina OV 1981 Tanycytes and their relation to the hypophyseal gonadotrophic function. Brain Research 210 253–260.[CrossRef][ISI][Medline]

Arias P, Carbone S, Szwarcfarb B, Feleder C, Rodriguez M, Scacchi P & Moguilevsky JA 1996 Effects of aging on N-methyl-D-aspartate (NMDA)-induced GnRH and LH release in female rats. Brain Research 740 234–238.[CrossRef][ISI][Medline]

Bestetti GE, Reymond MJ, Blanc F, Boujon CE, Furrer B & Rossi GL 1991 Functional and morphological changes in the hypothalamo-pituitary-gonadal axis of aged female rats. Biology of Reproduction 45 221–228.[Abstract]

Brawer JR & Walsh RJ 1982 Response of tanycytes to aging in the median eminence of the rat. American Journal of Anatomy 163 247–256.

Burger HG, Dudley EC, Robertson DM & Dennerstein L 2002 Hormonal changes in the menopause transition. Recent Progress in Hormone Research 57 257–275.[Abstract/Free Full Text]

Cashion AB, Smith MJ & Wise PM 2003 The morphometry of astrocytes in the rostral preoptic area exhibits a diurnal rhythm on proestrus: relationship to the luteinizing hormone surge and effects of age. Endocrinology 144 274–280.[Abstract/Free Full Text]

Clemens JA & Bennet DR 1977 Do aging changes in the preoptic area contribute to loss of cyclic endocrine function? Journal of Gerontology 32 19–24.[ISI][Medline]

Clemens JA, Tinsley FC & Fuller RW 1977 Evidence for a dopaminergic component in the series of neural events that lead to the proestrous surge of LH. Acta Endocrinologica (Copenhagen) 79 18–24.

Daftary SS & Gore AC 2003 Developmental changes in hypothalamic insulin-like growth factor-1: relationship to gonadotropin-releasing hormone neurons. Endocrinology 144 2034–2045.[Abstract/Free Full Text]

Daftary SS & Gore AC 2005 IGF-I in the brain as a regulator of reproductive neuroendocrine function. Experimental Biology and Medicine 230 292–306.[Abstract/Free Full Text]

Durrant AR & Plant TM 1999 A study of the gonadotropin releasing hormone neuronal network in the median eminence of the rhesus monkey (Macaca mulatta) using a post-embedding immunolabel-ling procedure. Journal of Neuroendocrinology 11 813–821.[CrossRef][ISI][Medline]

Estes KS & Simpkins JW 1982 Resumption of pulsatile luteinizing hormone release after alpha-adrenergic stimulation in aging constant estrous rat. Endocrinology 111 1778–1784.[ISI][Medline]

Flament-Durand J & Brion JP 1985 Tanycytes: morphology and functions: a review. International Review of Cytology 96 121–155.[ISI][Medline]

Funabashi T & Kimura F 1995 The number of luteinizing hormone-releasing hormone immunoreactive neurons is significantly decreased in the forebrain of old-aged female rats. Neuroscience Letters 189 85–88.[CrossRef][ISI][Medline]

Gerhold LM, Rosewell KL & Wise PM 2005 Suppression of vaso-active intestinal polypeptide in the suprachiasmatic nucleus leads to aging-like alterations in cAMP rhythms and activation of gonadotropin-releasing hormone neurons. Journal of Neuroscience 25 62–67.[Abstract/Free Full Text]

Gill S, Sharpless JL, Rado K & Hall JE 2002 Evidence that GnRH decreases with gonadal steroid feedback but increases with age in postmenopausal women. Journal of Clinical Endocrinology and Metabolism 87 2290–2296.[Abstract/Free Full Text]

Goldsmith PC & Ganong WF 1975 Ultrastructural localization of luteinizing hormone-releasing hormone in the median eminence of the rat. Brain Research 97 181–193.[CrossRef][ISI][Medline]

Gore AC & Roberts JL 1995 Regulation of gonadotropin-releasing hormone gene expression in the rat during the luteinizing hormone surge. Endocrinology 136 889–896.[Abstract]

Gore AC, Wu TJ, Rosenberg JJ & Roberts JL 1996 Gonadotropin-releasing hormone and NMDA-R1 gene expression and colocalization change during puberty in female rats. Journal of Neuroscience 16 5281–5289.[Abstract/Free Full Text]

Gore AC, Oung T, Yung S, Flagg RA & Woller MJ 2000a Neuroendocrine mechanisms for reproductive senescence in the female rat: gonadotropin-releasing hormone neurons. Endocrine 13 315–323.[CrossRef][ISI][Medline]

Gore AC, Yeung G, Morrison JH & Oung T 2000b Neuroendocrine aging in the female rat: The changing relationship of hypothalamic gonadotropin-releasing hormone neurons and N-methyl- D-aspartate receptors. Endocrinology 141 4757–4767.[Abstract/Free Full Text]

Gore AC 2002a GnRH: The Master Molecule of Reproduction. Norwell, MA: Kluwer Academic Publishers.

Gore AC 2002b Gonadotropin-releasing hormone (GnRH) neurons: gene expression and neuroanatomical studies. Progress in Brain Research 141 193–208.[Medline]

Gore AC, Oung T & Woller MJ 2002 Age-related changes in hypo-thalamic gonadotropin-releasing hormone and N-methyl-D-aspartate receptor gene expression, and their regulation by oestrogen, in the female rat. Journal of Neuroendocrinology 14 300–309.[CrossRef][ISI][Medline]

Gore AC, Windsor-Engnell BM & Terasawa E 2004 Menopausal increases in pulsatile gonadotropin-releasing hormone release in a nonhuman primate (Macaca mulatta). Endocrinology 145 4653–4659.[Abstract/Free Full Text]

Gudelsky GA & Porter JC 1981 Sex-related difference in the release of dopamine into hypophysial portal blood. Endocrinology 109 1394–1398.[Abstract]

Hall JE & Gill S 2001 Neuroendocrine aspects of aging in women. Endocrinology and Metabolism Clinics of North America 30 631–646.[CrossRef][ISI][Medline]

Hall JE, Lavoie HB, Marsh EE & Martin KA 2000 Decrease in gonadotropin-releasing hormone (GnRH) pulse frequency with aging in postmenopausal women. Journal of Clinical Endocrinology and Metabolism 85 1794–1800.[Abstract/Free Full Text]

Hoffman GE & Sladek JR Jr 1980 Age-related changes in dopamine, LHRH and somatostatin in the rat hypothalamus. Neurobiology of Aging 1 27–37.[CrossRef][ISI][Medline]

Hoffman GE & Finch CE 1986 LHRH neurons in the female C57BL/6J mouse brain during reproductive aging: no loss up to middle age. Neurobiology of Aging 7 45–48.[CrossRef][ISI][Medline]

Hoffman GE & Lyo D 2002 Anatomical markers of activity in neuro-endocrine systems: are we all ‘fos-ed out’? Journal of Neuroendocrinology 14 259–268.[CrossRef][ISI][Medline]

Hokfelt T 1973 Possible site of action of dopamine in the hypothalamic pituitary control. Acta Physiologica Scandinavica 89 606–608.[ISI][Medline]

Horvath TL, Cela V & van der Beek EM 1998 Gender-specific apposition between vasoactive intestinal peptide-containing axons and gonadotrophin-releasing hormone-producing neurons in the rat. Brain Research 795 277–281.[CrossRef][ISI][Medline]

Hrabovszky E, Turi GF, Kallo I & Liposits Z 2004 Expression of vesicular glutamate transporter-2 in gonadotropin-releasing hormone neurons of the adult male rat. Endocrinology 145 4018–4021.[Abstract/Free Full Text]

Huang HH & Meites J 1975 Reproductive capacity of aging female rats. Neuroendocrinology 17 289–295.[CrossRef][ISI][Medline]

Huang H-H, Marshall S & Meites J 1976 Capacity of old versus young female rats to secrete LH, FSH and prolactin. Biology of Reproduction 14 538–543.[Abstract]

Hwang C, Pu H-F, Hwang C-Y, Liu J-Y, Yao H-C, Tung Y-F & Wang PS 1990 Age-related differences in the release of luteinizing hormone and gonadotropin-releasing hormone in ovariectomized rats. Neuroendocrinology 52 127–132.[ISI][Medline]

Jennes L, Stumpf WE & Sheedy ME 1985 Ultrastructural characterization of gonadotropin-releasing hormone (GnRH)-producing neurons. Journal of Comparative Neurology 232 534–547.[CrossRef][ISI][Medline]

Kawakami SI, Hirunagi K, Ichikawa M, Tsukamura H & Maeda K-I 1998 Evidence for terminal regulation of GnRH release by excitatory amino acids in the median eminence in female rats: a dual immunoelectron microscopic study. Endocrinology 139 1458–1461.[Abstract/Free Full Text]

King JC & Rubin BS 1994 Dynamic changes in LHRH neurovascular terminals with various endocrine conditions in adults. Hormones and Behavior 28 349–356.[CrossRef][Medline]

King JC, Tobet SA, Snavely FL & Arimura AA 1982 LHRH immunopositive cells and their projections to the median eminence and organum vasculosum of the lamina terminalis. Journal of Comparative Neurology 209 287–300.[CrossRef][ISI][Medline]

Krajnak K, Kashon ML, Rosewell KL & Wise PM 1998 Aging alters the rhythmic expression of vasoactive intestinal polypeptide mRNA but not arginine vasopressin mRNA in the suprachiasmatic nuclei of female rats. Journal of Neuroscience 18 4767–4774.[Abstract/Free Full Text]

Kriegsfeld LJ, Silver R, Gore AC & Crews D 2002 Vasoactive intestinal polypeptide contacts on gonadotropin-releasing hormone neurons increase following puberty in female rats. Journal of Neuroendocrinology 14 685–690.[CrossRef][ISI][Medline]

Krohn PL 1955 Ovarian homotransplantation. Annals of the New York Academy of Sciences 59 443–447.[ISI][Medline]

Le W-W, Wise PM, Murphy AZ, Coolen LM & Hoffman GE 2001 Parallel declines in Fos activation of the medial anteroventral periventricular nucleus and LHRH neurons in middle-aged rats. Endocrinology 142 4976–4982.[Abstract/Free Full Text]

Levine JE 1997 New concepts of the neuroendocrine regulation of gonadotropin surges in rats. Biology of Reproduction 56 293–302.[Abstract]

Li S, Givalois L & Pelletier G 1997a Effects of aging and melatonin administration on gonadotropin-releasing hormones (GnRH) gene expression in the male and female rat. Peptides 18 1023–1028.[CrossRef][ISI][Medline]

Li S, Givalois L & Pelletier G 1997b Dehydroepiandrosterone administration reverses the inhibitory influence of aging on gonadotrophin-releasing hormone gene expression in the male and female rat brain. Endocrine 6 265–270.[ISI][Medline]

Liposits Z, Reid JJ, Negro-Vilar A & Merchenthaler I 1995 Sexual dimorphism in copackaging of luteinizing hormone-releasing hormone and galanin into neurosecretory vesicles of hypophysiotrophic neurons: estrogen dependency. Endocrinology 136 1987–1992.[Abstract]

Lloyd JM, Hoffman GE & Wise PM 1994 Decline in immediate early gene expression in gonadotropin-releasing hormone neurons during proestrus in regularly cycling, middle-aged rats. Endocrinology 134 1800–1805.[Abstract]

Meister B, Hokfelt T, Tsuruo Y, Hemmings H, Ouimet C, Greengard P & Goldstein M 1988 DARPP-32, a dopamine- and cyclic AMP-regulated phosphoprotein in tanycytes of the mediobasal hypothalamus: distribution and relation to dopamine and luteinizing hormone-releasing hormone neurons and other glial elements. Neuroscience 27 607–622.[CrossRef][ISI][Medline]

Merchenthaler I, Lengvari I, Horvath J & Setalo G 1980 Immunohistochemical study of the LHRH-synthesizing neuron system of aged female rats. Cell and Tissue Research 209 499–503.[ISI][Medline]

Miller BH & Gore AC 2001 Alterations in hypothalamic insulin-like growth factor-I and its associations with gonadotropin-releasing hormone neurons during reproductive development and ageing. Journal of Neuroendocrinology 13 728–736.[CrossRef][ISI][Medline]

Miller BH & Gore AC 2002 N-Methyl-D-aspartate receptor subunit expression in GnRH neurons changes during reproductive senescence in the female rat. Endocrinology 143 3568–3574.[Abstract/Free Full Text]

Miller MM & Zhu L 1995 Ovariectomy and age alter gonadotropin hormone releasing hormone-noradrenergic interactions. Neurobiology of Aging 16 613–625.[CrossRef][ISI][Medline]

Miller MM, Joshi D, Billiar RB & Nelson JF 1990 Loss of LH-RH neurons in the rostral forebrain of old female C57BL/6J mice. Neurobiology of Aging 11 217–221.[CrossRef][ISI][Medline]

Nozaki M, Mitsunaga F & Shimizu K 1995 Reproductive senescence in female Japanese monkeys (Macaca fuscata): age- and season-related changes in hypothalamic-pituitary-ovarian functions and fecundity rates. Biology of Reproduction 52 1250–1257.[Abstract]

Prevot V, Dutoit S, Croix D, Tramu G & Beauvillain JC 1998 Semi-quantitative ultrastructural analysis of the localization and neuropeptide content of gonadotropin releasing hormone nerve terminals in the median eminence throughout the estrous cycle of the rat. Neuroscience 84 177–191.[CrossRef][ISI][Medline]

Rance NE & Uswandi SV 1996 Gonadotropin-releasing hormone gene expression is increased in the medial basal hypothalamus of postmenopausal women. Journal of Clinical Endocrinology and Metabolism 81 3540–3546.[Abstract]

Rangaraju NS, Xu JF & Harris RB 1991 Pro-gonadotropin-releasing hormone protein is processed within hypothalamic neurosecretory granules. Neuroendocrinology 53 20–28.[ISI][Medline]

Reymond MJ & Porter JC 1981 Secretion of hypothalamic dopamine into pituitary stalk blood of aged female rats. Brain Research Bulletin 7 69–73.[CrossRef][ISI][Medline]

Romero M-T, Silverman A-J, Wise PM & Witkin JW 1994 Ultra-structural changes in gonadotropin-releasing hormone neurons as a function of age and ovariectomy in rats. Neuroscience 58 217–225.[CrossRef][ISI][Medline]

Rossmanith WG 1995 Gonadotropin secretion during aging in women: review article. Experimental Gerontology 30 369–381.[CrossRef][ISI][Medline]